Biological soil detector

ABSTRACT

A biological soil detector is provided, having: a solid support member; a specific indicator immobilized on the solid support member; and a retaining member which holds the solid support, the retaining member and solid support are configured so that the solid support will readily contact a surface being tested for the presence of biological soil. A method for the use of the detector is also provided, the method including the following steps: contacting the biological soil detector with a surface; withdrawing the biological soil detector from the surface; and inspecting the solid support member of the detector to determine whether the specific indicator immobilized has interacted with biological soil. In still another aspect, the invention provides biological soil detector and a method for using the detector in which the detector includes: a solid support member that has been treated with at least one amine or quaternary functional coating; and a specific indicator immobilized on the coating.

The present invention relates to a biological soil detector and a methodfor using a biological soil detector.

BACKGROUND OF THE INVENTION

The general ability to detect biological soil on any of a variety ofsurfaces is desired. In food preparation, either in the commercial orhome setting, the detection of biological soil on food preparationsurfaces and the like is valuable to prevent cross-contamination of fooditems being prepared on the surface. Examples of contaminating materialsmay include bacteria, food products that contain bacteria (e.g. raw meatand its juices), or certain biological fluids. To preventcross-contamination, it may be desirable to determine the level of soilon certain surfaces suspected of being in contact with biological soilsuch as kitchen and bathroom surfaces (e.g. counters, cutting boards,toilets), room surfaces (e.g. floors, walls) and the like. An assessmentof cleanliness is important for the surfaces of medical devices exposedto biological fluids during use. Examples of medical devices include thesurfaces of endoscopes, catheters, and other devices.

While kits are commercially available to test the cleanliness of certainsurfaces, available kits typically require samples to be sent to anoutside laboratory for analysis. The time involved in sending samples toan outside laboratory for analysis must be factored into the timerequired for the requester to receive a response. Additionally, culturemethods are typically employed in the analysis for pathogens, thusrequiring microbiology laboratory equipment and the expertise of trainedmicrobiologists.

In health care fields, medical devices such as endoscopes find utilityin medical procedures that expose the devices to biological soil.Endoscopes, for example, are used in medical procedures within apatient's body in which the endoscope is inserted into the body eitherthrough a natural orifice or through a surgical opening. Endoscopesinclude a number of channels that may carry optical fibers for viewingareas in the body to facilitate the examination of organs, joints orbody cavities and for conveying light to the area being viewed.Operating instruments such as electrosurgery probes or forceps may bepassed through the channels of an endoscope, and the channels may alsobe used to deliver fluids or gas, or to provide suction or pass samplingcatheters therethrough.

Virtually any part of the human body is accessible to an endoscope, andtypical surgical sites include the ear, throat, urinary tract, lungs,intestines and the abdominal cavity. Endoscopes used in colonoscopyprocedures permit the direct examination of the inside of the colon andlarge intestines for the presence of polyps, ulcers and inflammation.Foreign bodies such as polyps or tumors may be surgically removedthrough the endoscope.

As may be apparent, endoscopes are exposed to any of a variety of bodysoil during their use in surgical procedures. Such soils include blood,fecal matter, cellular matter from various tissue, and the like, and anyof these soils may provide sources of viruses or bacteria. Because oftheir use within the body, each endoscope must be thoroughly cleaned anddisinfected following each use to ensure that all of the soil-containingsurfaces are disinfected prior to using the medical device in subsequentmedical or surgical procedures. In one recommended cleaning processemployed on reusable endoscopes, the soiled endoscope is initiallycleaned during a manual cleaning step to remove as much soil as possiblefrom all of the soiled surfaces of the instrument. Thereafter, a highlevel disinfection step is performed on the manually cleaned endoscopeto render it ready for reuse. Typically, the manual cleaning step isperformed by scrubbing the instrument with a cleaning brush or similarinstrument. The manual cleaning step is performed until the brush nolonger appears to pick up soil from the surfaces of the instrument. Inthe absence of an effective manual cleaning process, bacterialcontamination may not be reduced to sufficiently low levels, thusincreasing the possibility that the subsequent high level disinfectionprocess may not be effective.

Currently, there is no standard test methodology that provides a rapiddetermination of the efficacy of the manual cleaning step.

The ability to evaluate the efficacy of a cleaning or disinfectingprocess for any of a variety of surfaces is desirable. It is desirableto provide a method that avoids extended incubation periods andfacilitates the rapid identification of the presence of certainbiological soil. It is also desirable to provide an article or devicethat can be used in the performance of the foregoing method.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a biological soildetector, comprising:

-   -   A solid support member;    -   A specific indicator immobilized on the solid support member;        and    -   A retaining member holding the solid support thereon, the        retaining member and solid support configured to facilitate        contact between the solid support and a surface.

In another aspect, the invention provides a method for detectingbiological soil associated with a surface, the method comprising:

-   -   Contacting the foregoing biological soil detector with a surface        wherein the contacting initiates the modification of the        specific indicator in the presence of bio soil;    -   Withdrawing the biological soil detector from the surface;    -   Inspecting the solid support member of the detector for a        detectable response to thereby determine whether the specific        indicator immobilized on the solid support has interacted with        biological soil.

In still another aspect, the invention provides a biological soildetector, comprising:

-   -   A solid support member treated with at least one amine or        quaternary functional coating; and    -   A specific indicator immobilized on the coating.

In still another aspect, the invention provides a method for detectingbiological soil associated with a surface, the method comprising:

-   -   Contacting the foregoing biological soil detector with a surface        wherein the contacting initiates the modification of the        specific indicator in the presence of bio soil;    -   Withdrawing the biological soil detector from the surface;    -   Inspecting the solid support member of the detector for a        detectable response to thereby determine whether the specific        indicator immobilized on the solid support has interacted with        biological soil.

As used herein, the terms used in the description of the variousembodiments of the invention will be understood to have their ordinaryand accustomed meaning unless stated otherwise. For convenience,specific definitions are provided for certain terms, such as thefollowing:

“Biological soil” or “bio soil” refer to, by way of example, body fluids(e.g., saliva, blood, digestive fluids) fecal matter, cellular materialsand tissue, microbial matter, bacteria, viruses, pathogens and otherbiological or biochemical materials including enzymes as well aspartially or wholly digested foods. Sources of biological soil may varybut may include blood, human bodies, animal bodies, plant matter andvarious food products such as meats, poultry, dairy products which may,for example, be contaminated or which are at least partially digestedand/or decomposed.

As used herein, “patient soil” refers to biological soil that remains ona medical device following the removal of the device from a human body.

A “specific indicator” refers to one or more chemical compounds thatwill interact with an enzyme or protein, such as those enzymes found inbiological soil, to thereby provide a detectable response such asvisible color changes or detectable changes in the fluorescent orluminescent properties of the specific indicator.

“Immobilized” refers to the retention of a chemical compound on a solidsubstrate in a manner that will resist removal of the compound from thesubstrate when the substrate is exposed to water.

Additional details of the preferred embodiments are provided in theremainder of the disclosure including the Detailed Description Of ThePreferred Embodiment and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

In the description of the preferred embodiment, reference is made to thevarious Figures wherein reference numerals are used to identify featuresof the depicted embodiments with like reference numerals indicating likestructures and wherein:

FIG. 1 a is a side elevation, in cross section, of a portion of oneembodiment of a solid support member according to the invention;

FIG. 1 b is a side elevation, in cross section, of a portion of anotherembodiment of a solid support member according to the invention;

FIG. 1 c is a side elevation, in cross section, of a portion of anotherembodiment of a solid support member according to the invention;

FIG. 2 is a perspective view representing an embodiment of a detectoraccording to the invention;

FIGS. 3-6 are perspective views of different embodiments of a feature ofthe embodiment depicted in FIG. 2;

FIG. 7 is a perspective view of an embodiment of a detector according tothe invention;

FIG. 8 is a perspective view of another embodiment of a detectoraccording to the invention;

FIG. 9 is a perspective view of another embodiment of a detectoraccording to the invention;

FIGS. 10-12 are views illustrating various embodiments of a retainingmember and a solid support member for a detector according to theinvention;

FIG. 13 a is a side elevation, showing another embodiment of a featureof the invention;

FIG. 13 b is a side elevation, in partial cross section, illustrating amethod of using the device of FIG. 13 a, according to the invention;

FIG. 14 shows another embodiment of a retaining member and a solidsupport member for a detector according to the invention;

FIG. 15 shows another embodiment of a retaining member and a solidsupport member for a detector according to the invention;

FIGS. 16-18 are various views of a feature of another embodiment of adetector according to the invention; and

FIGS. 19-21 are various views of a feature of another embodiment of adetector according to the invention.

FIGS. 22 a-22 c are various views of patterns that may be employed inembodiments of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention provides a detector and a method for the detectionof biological soil. The detector of the invention utilizes one or morespecific indicators immobilized on a solid support member wherein thespecific indicators provide a detectable response when contacted withbiological soil. The present invention generally relates to thedetection of markers or analytes (e.g., detectable biochemicalsubstances) that are indicative of the presence of biological soil.Detectable markers include components found in biological soil includingany of a variety of proteins or enzymes found in or originating from acomponent of biological soil. The detector and method of the inventionare suitable for use to determine the effectiveness of a cleaning stepperformed on a medical device. Additional uses for the device of theinvention are also contemplated, such as the detection of bio soil onother surfaces including those used for food preparation or processing,for example.

In one aspect, the invention provides a means for associating the biosoil on a surface, such as a surface on a medical device, with aspecific indicator immobilized on a solid support member. The specificindicator may be chosen for its sensitivity to components of the biosoil so that bio soil from a surface will react with the specificindicator to generate a detectable, relatively rapid, response.Detectable responses may be provided in the form of a color change onthe surface of the solid support member or by a change in thefluorescent properties of the specific indicator. In variousembodiments, the device of the invention includes the aforementionedsolid support member which may be used to directly contact the surfacebeing tested or may be indirectly used to test for the presence of biosoil by, for example, contacting the solid support member with a liquidthat has been used to rinse the surface being tested. Typically, thelatter embodiment will provide for the capture of the liquid within areceptacle that also holds the specific indicator immobilized on a solidsupport member so that the capture of the liquid will expose the solidsupport member and the immobilized specific indicator to the bio soilrinsed from the surface.

In some embodiments, the invention provides a means to determine thepresence of biological soil on a medical device. One such applicationfor the invention is the detection of biological soil following themanual cleaning step for an endoscope, for example. An endoscope permitsdirect viewing of areas within the body by insertion of the devicethrough a natural orifice or through a small incision in the skin. Someendoscopes are rigid structures employing a series of lenses, whileothers are flexible and employ optical fibers to illuminate the area ofconcern within the body and to convey an image back to the eyepiece forthe surgeon to see. Surgical operating instruments may be passed intothe body through the channels of the endoscope in order to performsurgical procedures such as electrosurgery or the manipulation, graspingor crushing of structures within the surgical area. Endoscope channelsmay also deliver fluids or gases into the surgical site, provide suctionor facilitating the positioning of catheters or laser light pipes. Inthe case of flexible endoscopes, an operating handle allows the surgeonto manipulate the tip of the endoscope to the desired location withinthe surgical site.

Following a use of an endoscope in a medical procedure, a manualcleaning process is employed to remove visible bio soil from the outersurface of the endoscope as well as from the inner surfaces or lumen ofeach exposed channel. After the manual cleaning step, the instrument maybe disinfected using an appropriate high level disinfectant. Theinvention provides a means for detecting the presence of residualbiological soil on an endoscope or other medical device to determinewhether a cleaning step was successful or whether detectable soil isstill present on the device so that the cleaning step must be repeated.If no soil is detected, the endoscope or other medical device isconsidered to be ready for high level disinfection.

Although the embodiments of the invention are typically described inconnection with their use in the detection of bio soil on endoscopes, itwill be appreciated that the invention is not to be limited to endoscopyapplications. The invention may also be used to test other medicaldevices as well as surfaces used for the preparation, examination and/ortreatment of patients in the healthcare industry. Additionally, theinvention can be used in any of a variety of industries outside of thehealthcare industry such as the food and beverage industry where theremay be a concern that a surface might become soiled due to inadequatecleaning or the like. Moreover, the invention is useful in the testingof surfaces in homes and offices including bathroom surfaces, kitchensurfaces and the like. In its various aspects, the invention is suitablefor rapidly testing any surface for the presence of a detectable amountof bio soil.

In at least one aspect, the invention provides a detector that includesa specific indicator with a means for contacting the specific indicatorwith a component of biological soil to produce a detectable response. Asis further described herein, the detector can be provided in any of avariety of embodiments wherein the specific indicator may be selected tosuitably detect biological soil and wherein the means for contacting thespecific indicator with a component of biological soil can also beprovided in any of a variety of forms. Embodiments of the invention andthe various components thereof are further illustrated and discussedbelow.

In the various embodiments of the invention, a solid support member isprovided along with a specific indicator immobilized on the solidsupport member. Referring to the various Figures, FIG. 1 a illustratesan embodiment of a solid support member 10 having a first surface 12 anda second surface 14. In the depicted embodiment, the support 10 is aporous material comprising open areas or pores 22, and solid portions 24extending between the pores 22. An optional backing 16 is affixed to thesecond surface 14 of the support 10. An optional scrim 18 extendsthrough the support 10 to provide additional strength for the support 10so that it can withstand significant stretching or pulling when, forexample, the support is pushed through a channel in a medical devicesuch as an endoscope. The scrim 18 may comprise any of a variety ofreinforcing materials including woven, nonwoven or knitted materials.

A specific indicator chemistry is immobilized on the solid supportmember 10 in at least one area of the support. In the embodiment shownin FIG. 1 a, the specific indicator chemistry is identified as the layer20 associated, in part, with the first surface 12. While the specificindicator layer 20 is mainly associated with the first surface 12, theindicator may extend through the body of the support 10 (as indicated bythe shaded portions of the support 10). It is contemplated that theindicator may be associated with the solid support member along a layeron a surface of the solid support member (e.g., layer 20), or theindicator may be associated in part with a surface of the solid supportmember, or the indicator may be mainly or completely disposed within thebody of the solid support member 10. As used herein, “immobilized on,”when referring to the placement of the specific indicator relative tothe solid support member, will be understood to encompass all possibleplacements of the specific indicator relative to the solid supportmember and is not intended to be limited to the placement of thespecific indicator at or on a surface of the solid support member.Additionally, more than one area of the solid support member may beassociated with a specific indicator and the use of multiple (e.g., twoor more chemically different) specific indicators on the same solidsupport member is contemplated within the scope of the invention.

Referring to FIG. 1 b, another solid support member 110 is illustratedin the form of a nonwoven material or fabric comprising an assembly offibers 122 which may be oriented in a single direction or in a randommanner. The nonwoven solid support member 110 may be held together inany manner known to those of skill in the art, including (1) bymechanical interlocking of the fibers 122; (2) by the fusing ofthermoplastic or binding fibers, or (3) by the adhesive bonding of thefibers with an appropriate binder such as a rubber, starch, glue,casein, latex, or a cellulose derivative or synthetic resin.

As in the embodiment of FIG. 1 a, the solid support member 110 of FIG. 1b includes a first surface 112 and a second surface 114. An optionalbacking 116 is affixed to the second surface 114 of the support 110. Anoptional scrim 118 extends through the support 110 to providereinforcement so that the support 110 is able to withstand thestretching or pulling expected during use such as when, for example, thesupport 110 is pushed through a channel in a medical device such as anendoscope. Specific indicator chemistry is immobilized on the solidsupport member 110 in at least one area of the support. In theembodiment shown in FIG. 1 b, the specific indicator chemistry isidentified as the layer 120 associated, in part, with the first surface112 but extending through the body of the support 110 (as indicated bythe shaded portions of the support 110). In other aspects, theembodiments of FIGS. 1 a and 1 b are substantially the same.

Referring to FIG. 1 c, another solid support member 210 is shown, havinga first surface 212 and a second surface 214. Optional backing 216 isaffixed to the second surface 214 of the support 210, and optional scrim218 extends through the support 210 to reinforce the support 210.Specific indicator chemistry is immobilized on the solid support member210 in at least one area of the support. In the embodiment shown in FIG.1 c, the specific indicator chemistry is identified as the layer 220associated, in part, with the first surface 212 but extending throughthe body of the support 210 (as indicated by the shaded portions of thesupport 210). The solid support member 210 may comprise any of a varietyof materials capable of having a specific indicator layer 220immobilized on the solid support member. Such materials are describedelsewhere herein. In all remaining aspects, the embodiments of FIGS. 1a, 1 b and 1 c are substantially the same.

The solid support member in the foregoing embodiments of FIGS. 1 athrough 1 c may be used, for example, as a single-use or disposable wipefor the detection of bio soil on various surfaces such as surfaces thathave been in contact with food (e.g., food preparation surfaces, food ormeat processing areas and the like), bathroom and kitchen sinks,counters, cutting boards, toilet surfaces, floors, walls, and any othersurface where biological soil may be present. Such a wipe may be useddry (e.g., on a wet surface), or it may be wetted with water or anaqueous solution. In some embodiments, a cleaning or disinfectingsolution may be incorporated into the wipe so that the cleaning ordisinfecting of a surface may be performed at the same time the surfaceis being tested for bio soil. Accordingly, the invention provides a wipethat is useful in the evaluation of the cleanliness of a surface so thatadditional cleaning or disinfecting may be performed on the surface ifthe specific indicator interacts with a component of patient soil toprovide an observable color change on the surface of the wipe or achange in the fluorescence of the surface of the wipe.

As noted, the solid support member is a substrate for the immobilizationof a specific indicator. Suitable materials for the solid support membermay be either a single base material having desirable surfacecharacteristics, or a composite structure. If the solid support memberis a single base material, suitable materials are polymers, inorganic,or mixed organic and inorganic surfaces that exhibit a contact anglewith water of less than 90 degrees, preferably less than 50 degrees, andmost preferably less than 10 degrees. Suitable materials include, butare not limited to, polymers containing the following functional groups:carboxyl groups and salts thereof, aldehydes, sulfonic acid and saltsthereof, phosphonic acid and salts thereof, alcohol, primary amine,secondary amine, tertiary amine, amide, imide, quaternized ammonium,sulfonium, phosphonium, pyridine, cyclic amido (e.g. 2-pyrrolidinonyl,2-piperidinonyl), oxyalkylene, and imidazole. Polymers or copolymersthat contain or may be prepared to contain these functional groupsinclude, but are not limited to, the following: carboxyl containingpolymers such as, e.g., polymers and copolymers synthesized from acrylicacid and/or methacrylic acid including salts thereof; polyalkoxylates;poly(meth)acrylates; polyvinyl alcohol and copolymers, such aspolyethylene-vinyl alcohol copolymer (e.g., available under the tradedesignation EVAL F101A from EVAL Company of America (EVALCA), Houston,Tex.); polyurethanes; polyureas; polyesters; polyamides, such as nylon6,6; polyimides; polyethers; celluloses such as cellulose acetate,nitrocellulose, hydroxymethylcellulose, hydroxypropylcellulose; rayon;polyphosphate; polypeptide; polyacrylonitrile; polyacrylamide;polycarbonate; polyethersulfone; and combinations thereof.

Suitable inorganic materials include metal oxides, hydrates, andmetal-hydroxyls (e.g. silicon hydroxyl (Si—OH) functional surfaces).Materials of construction that are both mixed organic and inorganicmaterials and suitable as supports include, but are not limited to,polymeric composites and ceramers, such as those based oncopolymerization of metal alkoxides (e.g. tetraethoxyorthosilicate,n-hydroxypropyltrimethoxysilane) and organic monomers.

In embodiments where the solid support member is a composite structure,the first material or base material may be any polymeric, inorganic, ormixed organic and inorganic material to which the second material orcoating material having desirable surface characteristics will adhere.Suitable base materials include, but are not limited to, polypropylene,polyethylene, polyvinylidene fluoride (PVDF), tetrafluoroethylenehexafluoropropylene vinylidene fluoride (THV), polyurethane, polyurea,polyester, polyvinyl acetate, polyamides, polyimides,poly(meth)acrylates, polyethersulfone, glass, silica, cellulosics,rayon, polycarbonate, polyvinyl alcohol, polystyrene, and combinationsof the foregoing.

The base material may be modified via the application of suitablecoating materials or surface treatments known to those skilled in theart to prepare a surface having a contact angle of water of less than 90degrees, generally less than 50 degrees, and typically less than 10degrees. Suitable coating materials may be prepared from monomers,polymers, or reactive metal alkoxides that may include one or morefunctional groups such as carboxylic acids and salts thereof, aldehydes,sulfonic acids and salts thereof, phosphonic acids and salts thereof,alcohols, primary amines, secondary amines, tertiary amines, amides,imides, quaternized ammonium, sulfonium, phosphonium, pyridine, cyclicamido groups (e.g. 2-pyrrolidinonyl, 2-piperidinonyl), oxyalkylene,ω-saccharinamidoundecylsiloxane (such as is described in Example 11 ofU.S. patent application Ser. No. 10/713,174 filed Nov. 14, 2003),glycidyl, succinimido groups and imidazoles. Coating materials also maybe prepared from monomers, polymers, or reactive metal alkoxides that donot contain functional groups including, but not limited to, alcohols,aldehydes, carboxylic acids, sulfonium, and phosphonium, but that may besubsequently modified by chemical reaction (e.g. oxidation, hydrolysis,degradation) to expose those groups at the surface. Coating materialsmay be applied using any known coating method including pattern coating(e.g. the coating material may be dropped in spots onto the basematerial). Surface treatment methods for preparation of a coatingmaterial suitable for a solid support member include but are not limitedto: oxygen plasma, corona treatment, flame treatment, chemical vapordeposition, graft polymerization, and physical vapor deposition. As usedherein, the term “coating” will be understood to include allconstructions wherein a second material is applied to a first materialon a solid support member such as continuous coatings, discontinuouscoatings, coatings applied or arranged in a discontinuous pattern,coatings applied in a continuous pattern but arranged in a geometric ora non-geometric configuration, and the like.

The solid support member can be made to comprise materials that include:films, nonwoven materials such as cellulosic materials and materialsthat include a rayon/polypropylene nonwoven materials (e.g., thoseavailable under the trade designation Novonette 149-051 from BBANonwovens, Nashville, Tenn.) and nonwoven materials comprising rayon andpolyester (e.g., 70% rayon/30% polyester), woven or knitted materials(e.g., prepared from cotton, rayon, or polymer materials), reticulatedfoams (e.g., polyurethane), open-celled foams (e.g., (meth)acrylate,polystyrene divinybenzene), porous ceramic inorganic frits (e.g.,silica, alumina), fibers, particle-coated supports, sintered particles,sintered fibers, sponges (e.g., arranged in a brush like configuration),fiber bundles and membranes. In some embodiments of the invention, suchas those to be inserted within a channel of a medical device (e.g., anendoscope), the solid support member comprises conformable, flexible,high integrity materials that are able to conform to and fit within theinner channels of a medical device while maintaining contact with theinner surfaces of the channel without experiencing structural failure(e.g., tearing or leaving remnants within the channel) when the solidsupport member is pushed and/or pulled through the length of thechannel.

Suitable polymer membranes for use as the solid support member includethose resulting from a phase inversion method in which an initiallyhomogeneous polymer solution is cast and exposed to a cooler interface(e.g., a water bath or chilled casting wheel), and phase separation isinduced in the solution film by lowering the temperature (thermallyinduced phase separation or “TIPS”). Suitable TIPS films or membranesmay possess a broad range of physical film properties and microscopicpore sizes. They may be relatively rigid or non-rigid substratesprepared from any of a variety of polymers. TIPS membranes madeaccording to the teachings of U.S. Pat. Nos. 4,539,256 and 5,120,594 aresuitable for use in the invention and may comprise high densitypolyethylene (HDPE), polypropylene, polyvinylidenefluoride (PVDF),polyethylene-vinyl alcohol copolymer (e.g., available under the tradedesignation EVAL F101A from EVAL Company of America (EVALCA), Houston,Tex.), for example. The membrane may comprise a combination of materialssuch as a TIPS HDPE or a polypropylene membrane coated with ahydrophilic polymer (e.g., polyethylene-vinyl alcohol copolymer or EVAL)or a TIPS polypropylene support coated with a hydrophilic, stronglybasic positively-charged coating such as polydiallyldimethylammoniumchloride or a polymer incorporating quaternizeddimethylaminoethylacrylate. The membrane also may comprise a stronglybasic, positively-charged membrane comprising polyethersulfone copolymerwith quaternary ammonium groups such as a membrane commerciallyavailable from Pall Corporation of Pensacola, Fla. under the tradedesignation “SB-6407.” Other supports may comprise nonwoven materialsprepared from non-rigid polymers and other materials including nylonmaterials such as positively charged Nylon 6,6 materials (e.g., thoseavailable under the trade designation Biodyne B from Pall Corporation,Pensacola, Fla. and those available under the trade designationMagnaprobe from GE Osmonics Labstore in Minnetonka, Minn.), ahydrophilic treated polypropylene membrane with 0.45 micron pore size,available under the trade designation GHP-450 from Pall Corporation,polyolefins (with a hydrophilic treatment); polyester, nitrocellulose,cellulose acetate, hydrophilic polytetrafluoroethylene (PTFE),polycarbonate, and the like. Combinations of materials may be used as asolid support member and the foregoing description is to be understoodto include the aforementioned materials alone and in combination withother materials.

Regarding specific indicators, compounds suitable for use as specificindicators may be selected from any of a variety of materials capable ofinteracting with a component of bio soil to provide a detectableresponse. A consideration in the selection of a specific indicator is toselect an indicator that will not react with cleaning solutions orcomponents thereof or other substances that do not originate frombiological soil, such as those substances that are introduced during themanual cleaning step for a medical device such as an endoscope.Individual compounds may be used as a specific indicator as well ascombinations of compounds. Suitable specific indicators include, forexample, 5-bromo-4-chloro-3-indolyl β-D-glucopyranoside;5-bromo-4-chloro-3-indolyl β-D-galactopyranoside;5-bromo-4-chloro-3-indolyl phosphate;5-bromo-6-chloro-3-indolyl-β-D-glucopyranoside;5-bromo-6-chloro-3-indolyl-β-D-galactopyranoside;5-bromo-6-chloro-3-indolyl phosphate;4-methylumbelliferyl-β-D-glucopyranoside;4-methylumbelliferyl-β-D-galactopyranoside;4-methylumbelliferyl-phosphate, esculin, orthophthaldialdehyde (OPA),polydiacetylenes as described in U.S. Pat. Nos. 6,395,561B1;6,306,5989B1; 6,277,652; 6,183,722; 6,080,423 and WO 01/71317; Bradfordassay based on the binding of Coomassie Brilliant Blue dye to proteins(available from Pierce Biotechnology Inc. of Rockford, Ill.); Lowryassay based on the reduction of the phosphomolybdic-tungstic mixed acidchromogen by a protein; Biuret assay based on the interaction of Cu+2with protein in an alkaline solution; and the bicinchoninic acid (BCA)(available from Pierce Biotechnology Inc. of Rockford, Ill.) to detectthe reduction of Cu+2 ions to Cu+1 in the presence of protein.Combinations of two or more of the foregoing immobilized on a solidsupport member are also contemplated within the scope of the invention.Additionally, when indolyl functional indicators are used in combinationwith nitro blue tetrazolium chloride (NBT) or other electron acceptors,faster development of color will occur in the presence of biologicalsoil.

Enzyme activity maybe enhanced by the addition of monovalent or divalentmetal ions, e.g., sodium, potassium, zinc, manganese, magnesium,calcium. Manganese salts can be incorporated in indicator formulationsthat include NBT to avoid the premature development of color (e.g., inthe absence of bio soil).

In embodiments where the detector is provided as a wipe, the specificindicator typically comprises 5-bromo-4-chloro-3-indolylβ-D-glucopyranoside; 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside;5-bromo-4-chloro-3-indolyl phosphate;5-bromo-6-chloro-3-indolyl-β-D-glucopyranoside;5-bromo-6-chloro-3-indolyl-β-D-galactopyranoside;5-bromo-6-chloro-3-indolyl phosphate;4-methylumbelliferyl-β-D-glucopyranoside;4-methylumbelliferyl-β-D-galactopyranoside and combinations of two ormore of the foregoing. Nitro blue tetrazolium chloride (NBT) or otherelectron acceptors may be added to the foregoing specific indicators forfaster development of color in the presence of biological soil.

Enzyme activity maybe enhanced by the addition of monovalent or divalentmetal ions, e.g., sodium, potassium, zinc, manganese, magnesium,calcium. Manganese salts can be incorporated in indicator formulationsthat include NBT to avoid the premature development of color (e.g., inthe absence of bio soil).

A number of different means of immobilizing the specific indicator tothe support may be utilized; for example, adsorption, ion exchange,entrapment, microencapsulation, cross-linking, copolymerisation,entrapment and cross-linking, compounding, and covalent attachment.Adsorption of the indicator to the support occurs as a result of van derWaals, electrostatic and/or hydrophobic interactions between theindicator and support. Ion exchange results in binding of the indicatorto the support due to electrostatic attraction between charges on theindicator and support. Entrapment implies mechanical capture of theindicator inside microscopic or macroscopic voids in the support.Microencapsulation or encapsulation involves covering the indicatorchemistry either with a chemically different coating, usually for thepurpose of protecting the indicator from external environments untilexposure to a triggering physical or chemical event (e.g. sudden changein local relative humidity). Indicators can be crosslinked onto or intothe support if they have reactive groups attached to them, either viasurface-grafting or copolymerization into the bulk of the support, orthey may be entrapped and subsequently cross-linked into the support.Indicators also may be compounded as additives into polymeric supportsvia extrusion processing. Covalent attachment of indicators to thesupport may be achieved with solid support members functionalized withone or more ligands that react with functional groups on the indicator.Exemplary ligands include those mentioned in the Examples herein. It isdesirable that the immobilization is accomplished in a manner thatavoids steric hindrance in the reaction between the solid-phaseindicator and the solution-phase reactant. Additionally, theimmobilization should not inactivate the indicator. Particularly usefuland convenient techniques are entrapment of the indicator chemistry in amicroporous membrane and/or adsorption of the indicator chemistry to asupport.

The selection of specific indicator may be influenced by the markerspresent in biological soil. Exemplary markers for detection includeproteins, endotoxins, enzymes, and nucleotides such as adenosinetriphosphate (ATP). Protein is a useful marker for the presence ofbiological soil because of its ubiquitous presence in human secretionsas well as in microbial cell components. The presence of an endotoxinwould be representative of the lipopolysaccharide component ofgram-negative bacteria. Detection of enzyme activity would signify thepresence of enzymes that could be mammalian, plant or microbial inorigin. Suitable enzymes for detection include, without limitation,galactosidases, phosphatases, glucosidases, lactosidases and others thatare normally found in human secretions as well as those originating froma microbial or plant source. Other markers include sulfatases and fattyacid esterases.

Referring to FIGS. 7-10, a detector 310 according to the invention isshown wherein each of the Figures incorporate different features whichwill now be described. The detector 310 includes a solid support member312 associated with a retaining member 314 for retaining the solidsupport member 312 thereon. The retaining member 314 includes a firstend 316 and a second end 318 and an elongate body portion 320 extendingbetween the first end 316 and the second end 318. In the embodiment ofFIG. 7, the solid support member is affixed to the first end 316 of theretaining member 314. Additionally, the length of the retaining member314 may be varied as desired and is typically dimensioned in order toserve as a handle to facilitate contact between a specific indicatorimmobilized on the solid support member 312 and a surface to be tested.In this construction, the solid support member 312 may be positioned orextended to reach surfaces that may be out of reach or otherwiseinaccessible. In other words, the user of the detector 310 may grasp theend of the retaining member 314 that is distal to the solid supportmember (e.g., second end 318) and use the length of the elongate bodyportion 320 of the retaining member 314 to reach a relatively remote orinaccessible surface with the solid support member and the specificindicator associated therewith. In some embodiments, the retainingmember is dimensioned to fit within a channel of a medical instrument,such as a channel of an endoscope, to allow the solid support member 312and the specific indicator to sample the channel walls for the presenceof bio soil, including patient soil.

Referring to FIG. 8, the detector 310 includes solid support member 312affixed to the second end 318 of the retaining member 314. Also, acleaning brush 322 is provided and is affixed to the first end 316 ofthe retaining member 314. In this construction, the cleaning brush 322provides a means for cleaning. In some embodiments, the brush 322 issized and configured to clean a medical device such as an endoscope, andtypically the brush 322 is sized and configured for cleaning within achannel of a medical device. Following a cleaning operation, thedetector 310 may then be used to test for the remaining presence of biosoil on the device and especially within the channel(s) of the device.

In the foregoing embodiments, the retaining member is typically affixedto the solid support member in a permanent or non-removable manner. Inother words, the attachment between the solid support member and theretaining member is not generally intended to permit detachment of theseparts from one another. Regarding the manner of attachment, the solidsupport member and the retaining member may be attached to one anotherin any manner known to those of skill in the art including, withoutlimitation, adhesive attachment, melt bonding, mechanical attachment(e.g., staples, buttons, snaps or the like). It is contemplated that allmanners of attaching the solid support member to the retaining memberare within the skill of those practicing in the field are encompassedwithin the present disclosure.

FIG. 9 shows still another configuration for the detector 310 whereinthe brush 322 is associated with the first end 316 of the retainingmember 314. The solid support member 312 is shown detached from theretaining member 314 to illustrate that the solid support member 312 maybe releasably associated with the retaining member 314. In other words,the solid support member is provided as a separate component of thedetector 310 that is detached from the retaining member 312 but whichmay be affixed to the retaining member 314 at any place along the lengthof the elongate body portion 320 or at either of the first end 316 orthe second end 318. Moreover, the solid support member may be removedfrom the retaining member 314 following use so that the retaining member314 and the brush 322 may be cleaned, disinfected and possiblysterilized as needed for use in a subsequent application. In thedepicted configuration, the solid support member 312 includes wing-likeprojections 313 a and 313 b at an end of the solid support member. Thewing-like projections 313 a and 313 b may comprise an inner reinforcingstructure with a thin steel or metal wire to give each of theprojections 313 a and 313 b some reinforcement and to provide a meansfor the solid support member 312 to be affixed to the retaining member314 (e.g., by hand twisting of the wing-like projections around theouter circumference of the retaining member 314). Alternatively, thewing-like projections 313 a and 313 b may each be of a length to permitthe two projections to be fastened to each other and the retainingmember 314. It will be appreciated that the solid support member 312 andthe retaining member 314 may be releasably affixed to one another by anyknown means (e.g., by use of a clip, adhesive or other fastener).

In the configuration of the detector shown in FIG. 9, the detector 310may be provided with a single retaining member 314 and multiple solidsupport members 312 that have been pretreated with one or more specificindicators. In such an arrangement, the multiple solid support members312 may include the same specific indicator(s) or different specificindicator(s).

FIGS. 10-13 a illustrate alternate configurations for the solid supportmember in the detector of the invention. FIG. 11 depicts a solid supportmember 360 positioned along retaining member 364. The solid supportmember 360 is comprised of a plurality of bristle-like members 362projecting perpendicularly from the retaining member 364. Inapplications such as the detection of bio soil in a channel of a medicaldevice, a sufficient number of such bristle-like members 362 areprovided to ensure that the solid support member 360 samplessubstantially the entire surface of the channel when positioned therein.

FIG. 11 depicts another configuration of a solid support member 370useful in the present invention. The support member is configured topermit bidirectional use in sampling the inner channel of an endoscope.In other words, the solid support member 370 may be pushed and/or pulledthrough the inner channel of a medical device in determining thepresence or absence of detectable bio soil. Additionally, the solidsupport member 370 includes a relatively large surface area whichfacilitates the visual identification of a color change in the presenceof bio soil.

FIG. 12 depicts another configuration of a solid support member 380useful in the present invention. As shown, the solid support member 380may be made from a foam material that is configured to be directedthrough the inner channel of a medical device in a single direction. Thesolid support member 380 may be made using less material than, forexample, the solid support member 370 in the embodiment of FIG. 11.However, the solid support member 380 includes a sufficient surface areato facilitate a rapid and relatively easy identification of a colorchange caused by the interaction of bio soil and the specific indicatorassociated with the solid support member.

FIG. 13 a depicts another configuration of a detector 388 having a solidsupport member 390 that is flag-shaped comprising somewhat triangularshaped surfaces. The solid support member 390 is affixed to theretaining member 392 and comprises a first edge 394 attached to theretaining member and a second edge 396 remote from the first edge 394.The second edge 396 is shorter than the first edge 394 so that the sideedges 395 and 397 extend between the first and second edges 394 and 394in a non-parallel manner. Moreover, the angle (designated as α) formedbetween side edge 395 and the retaining member 392 is depicted as beingabout 45 degrees. In some embodiments, the angle may be different thanis shown but will typically be less than 90 degrees, more typically lessthan 45 degrees, and often between about 20 and about 45 degrees. Inthis configuration, the solid support member 390 is useful in thesampling of a channel within a medical device. The angle α between thesolid support member 390 and the retaining member 392 facilitate auniform wrapping of the solid support member 390 around the retainingmember 392 when inserted within the channel of a medical device, forexample. It will also be appreciated that the solid support member 390may be provided in a configuration in which the solid support member 390is wrapped around the retaining member 392. In such a configuration, thesize of the angle between side edge 395 and the retaining member 392becomes less important in the overall performance of the detector 388.

FIG. 13 b illustrates a use of the detector 388 in the sampling of thewall of a channel 400 in a medical device such as an endoscope. Thesolid support member 390 is of a length that facilitates the curling orwrapping of the solid support member in a ‘cork-screw’ or spiral patternaround the retaining member 392. The spiral wrapping of the solidsupport member 390 facilitates contact between a surface of the solidsupport member 390 and the channel walls along the entire channelsurface as the detector 388 is moved through the channel 400 in thedirection indicated by the arrow. In some embodiments, the length of thesolid support member is about 50 mm.

Referring now to FIG. 14, a solid support member 420 is depicted asaffixed to a retaining member 422, according to the invention. Solidsupport member 420 comprises a first area 424 comprised of a firstmaterial and a second area 426 comprised of a second material. Suitablefirst and second materials include those described elsewhere herein. Insome embodiments, the first material can comprise a nylon non-wovensheet, for example, and the second material can comprise a plurality ofTIPS membrane segments affixed to the first material. In someembodiments, the first and second materials are affixed to one anotherusing a hot melt adhesive applied (e.g., sprayed) to the TIPS segments.In this construction, a specific indicator may be immobilized within thesecond area 426. In embodiments where the specific indicator iscolorimetric, the detection of bio soil can be enhanced if the firstmaterial is chosen to provide a background color that will visiblyenhance the color contrast between the first and second materials upon acolor change in the second material 426 caused by the interaction of thespecific indicator with the bio soil.

Referring now to FIG. 15, a solid support 430 is depicted as affixed toa retaining member 432, according to the invention. Similar to theembodiment depicted in FIG. 14, solid support member 430 comprises afirst area 434 comprised of a first material and a second area 436comprised of a second material. In this construction, specific indicatormay be immobilized within the second area 436 such as by printing thespecific indicator on the solid support member within the second area436. In embodiments where the specific indicator is colorimetric, thedetection of bio soil can be enhanced if the first material is chosen toprovide a background color that will visibly enhance the color contrastbetween the first and second materials upon a color change in the secondmaterial 436 caused by the interaction of the indicator with the biosoil.

In the detector constructions of the invention, a solution of thespecific indicator may be coated onto the solid support member. Thesolvent may then be evaporated, thereby leaving the indicator compoundimmobilized on the solid support member. In some embodiments, the solidsupport member may be porous so that the specific indicator is retainedwithin the pores of the solid support member through physical entrapmentof the specific indicator within the pores, or by, for example, van derWaals forces, by hydrophobic and/or ionic interactions with the materialused in the solid support member. In some embodiments, the solid supportmember may be treated prior to applying the specific indicator in orderto render the solid support member hydrophilic and/or capable ofcovalently bonding with the specific indicator compound. A solution ofthe specific indicator may be coated uniformly over the entire surfaceof the solid support member or it may be coated onto the surface in apattern covering some portion of the surface.

In some embodiments, the surface of the solid support member may beprovided as uniformly white with the specific indicator immobilized onat least a portion of the white surface. In these embodiments, the whitebackground will provide a sharp color contrast to the color generated bythe reaction between the specific indicator and the bio soil and furtherfacilitating the identification of a colorimetric reaction. In someembodiments, the surface of the solid support member may initiallycomprise a low fluorescence background to facilitate detection of achange in fluorescence upon a reaction between the specific indicatorand bio soil.

In some embodiments, detection of a reaction between the indicatorcompound and biological soil may be enhanced if the indicator compoundis applied to the surface of the support in a predetermined or orderedpattern. Moreover, background patterns on the surface of the solidsupport member may be provided to enhance or emphasize the presence of acolorimetric reaction, such as the patterns shown in FIGS. 22 a-22 c. Ineach of the Figures, moving from left to right, a progression isillustrated showing a change from an initial pattern to a final patternfacilitated by a colorimetric reaction. Referring to FIG. 22 a, a seriesof small, closely grouped, darkly colored background circles 460 areprovided along with a second pattern comprised of non-colored or lightlycolored foreground circles 462. The foreground circles 462 may comprisevisible circles of a lighter color than the background circles 460 orthe foreground circles 462 may represent a colorless pattern of aspecific indicator immobilized on the surface of a solid support member.Upon the detection of biological soil, a calorimetric reaction causedthe foreground circles 462 to become visibly darker, so that the surfaceof the associated solid support member takes on a different appearanceas the colorimetric reaction progresses to completion. In the patternshown in FIG. 22 a, the series of diagonally extending broken linesappears to become a series of unbroken lines occupying a rectangulararea on the surface of the solid support member.

Similarly, FIG. 22 b illustrates a pattern of foreground circles or dots466 and background circles or dots 468 which progress into a geometricpattern as a colorimetric reaction progresses to completion. FIG. 22 cillustrates a series or pattern of background circles 472 which may beinitially presented as a lightly colored pattern or as a colorless andundetectable surface treatment applied to the solid support member. Aprinted or fixed mark, shown as a line segment 474 is provided over thebackground circles 472. As a colorimetric reaction progresses, thebackground circles become visibly darker until the appearance of thebackground circles 474 becomes the visually dominant feature, maskingthe presence of the segment 474. Patterns such as the foregoing arecontemplated within the scope of the invention as a possible form forpresenting the specific indicator on the solid support member tofacilitate the visual recognition of a colorimetric reaction indicativeof the presence of bio soil on a surface.

In embodiments of the invention, patterns like those described inrelation to FIGS. 22 a through 22 c, are imprinted or otherwise affixedto the solid support member. The pattern represents an area on the solidsupport member that is to be visually inspected for a calorimetricresponse following the sampling of a surface. Typically, the area of thepattern will be large enough to facilitate the easy and quick visualidentification of such a reaction, but the invention is not limited toany particular dimension or size of the area on the solid support memberthat has been treated with the specific indicator, whether in a patternor otherwise.

As mentioned, the detector of the invention is useful in the detectionof bio soil on a surface such as the surface of a medical device or anyother surface which may have been exposed to bio soil such as foodpreparation surfaces, for example. To detect for the presence of biosoil on a surface, the detector described above with reference to FIGS.7-14 may be used in a method comprising:

-   -   contacting the solid support member and the specific indicator        with the surface;    -   withdrawing the solid support member from the surface; and    -   inspecting the solid support of the detector for a detectable        response to thereby determine whether the specific indicator        immobilized on the solid support has interacted with a component        of biological soil.

In the foregoing method, the detector may be handled by a user graspingthe detector along the axis of the retaining member (e.g., retainingmember 314, FIG. 7), and extending the solid support member to aposition on the surface being examined so that the solid support memberand the specific indicator are in contact with the surface. Typically,the surface being tested and/or the solid support member will be atleast slightly wet (e.g., with water). Contact with the surface atambient temperature is generally sufficient to initiate an interactionbetween the bio soil (if present) and the specific indicator. When thedetector is withdrawn from the surface, the solid support member may beinspected for a change in appearance such as a color change. In someembodiments, the solid support member is examined to detect changes inthe fluorescence of the solid support member. Changes in color,appearance or fluorescence are indicative of the presence of bio soil.Typically, a change in appearance or fluorescence will be detectable inthe solid support member in a relatively short period of time.Generally, a detectable change will be observed within 15 minutes orless, more typically less than 10 minutes, even more typically, lessthan 5 minutes and often less than one minute. As shown in the appendedExamples, a detectable change is often apparent on the solid supportmember within 30 seconds or less. Additionally, the solid support memberretains the specific indicator prior to any contact with biological soiland also retains the reaction products resulting from the interactionbetween the specific indicator and bio soil.

The detector of the invention may be used for the detection of bio soilon any surface. Exemplary of such as surfaces are those that contactfood such as food preparation areas, food processing areas and the like.Any surface that potentially includes bio soil can be sampled with thedetector of the invention such as bathroom and kitchen surfaces (e.g.,sinks, counters, cutting boards, and toilet surfaces), floors and wallsor the like. In an aspect of the foregoing embodiment, a cleaning ordisinfecting solution may be incorporated into the solid support memberso that the cleaning or disinfecting of a surface may be performed atthe same time the surface is being tested for bio soil.

In some embodiments, the detector is useful for the detection of biosoil in the inner channels of a medical device. In the reprocessing ofreusable endoscopes, for example, the detector of the invention isuseful in the determination of the presence of residual bio soilfollowing the cleaning steps normally employed for endoscopereprocessing. In particular, a soiled endoscope that has been used in amedical or surgical procedure is typically cleaned by an initial manualcleaning step to remove visible debris or bio soil from the surfaces ofthe instrument, including the surfaces of the inner channels. This istypically accomplished with a brush and a cleaning solution or solventcomprising an enzymatic cleaning compound applied to the surfaces of theendoscope. The surfaces of the endoscope are normally scrubbed with abrush to remove all of the visibly detectable bio soil from the surfacesof the medical device. Thereafter, the endoscope is subjected to adisinfection step using a high level disinfectant. After disinfection ofthe device with the high level disinfectant, the endoscope is thoroughlyrinsed and dried so that it may be used again.

In order to evaluate the effectiveness of aforementioned manual cleaningstep, the detector of the invention described above with reference toFIGS. 7-14 may be used to determine whether detectable bio soil remainson any of the surfaces of the endoscope prior to subjecting theendoscope to high level disinfection. In such a method, the detector ofthe present invention is used by first contacting the solid supportmember with a surface. In some embodiments, the retaining member of thedetector is dimensioned to fit within the channels of the endoscope orother medical device to place the solid support member in contact withthe walls of the channel. When used within a channel, the solid supportmember is typically chosen from very flexible materials so that thesolid support fits within the channel in a manner facilitating contactbetween the specific indicator associated with the solid support memberand the channel walls. The retaining member is normally of a sufficientlength to ensure that the solid support member can be exposed to thewall of the channel along the entire length of the channel. Sampling ofa channel wall in a medical device will typically be accomplished bysliding the detector through the channel in a single direction.

In another aspect of the invention, the detector is provided in a formthat facilitates the detection of bio soil on the solid support memberby rinsing the surface being tested with water or another liquid andthereafter capturing the rinse fluid in a manner that facilitatescontact between the rinse fluid and the immobilized specific indicatoron a solid support member.

Referring now to FIG. 2, another embodiment of a detector is depictedaccording to the invention and will now be described. An endoscope 500is shown wherein one of its inner channels is being tested for thepresence of bio soil. At one end 502 of the endoscope 500, a suitablesampling or rinsing fluid 508 (e.g., water, phosphate buffered saline)is inserted into the channel of the endoscope 500 in a manner thatfacilitates the sampling of the entire length of the channel so that thefluid 508 exits the channel of the endoscope 500 at a second end 504thereof. The sampling fluid 508 is dispensed into the channel of theendoscope 500 with an appropriate amount of force to push the fluidthrough the channel. In some embodiments, a fluid dispenser such as asyringe 506 is used to dispense a predetermined amount of sampling fluid508 into the channel of the endoscope 500. The sampling fluid 508 exitsthe second end 504 of the endoscope 500 and contacts the solid supportmember 510 with the specific indicator immobilized within an area 512 ofthe solid support member 510.

It will be appreciated that the invention is not to be limited to thesampling of an endoscope. Sampling fluid can be applied to any of avariety of surfaces and subsequently brought into contact with aspecific indicator immobilized on a solid support member. The samplingfluid liquid may be brought into contact with the solid support memberin any manner such as by the placement of the solid support memberwithin the path of the sampling fluid as the fluid exits the channel orother surface being sampled. In some embodiments, the solid supportmember is simply placed within a stream of sampling fluid.

In some embodiments, the solid support member 512 is positioned within areceptacle that can also capture the sampling fluid 508 after the fluidhas contacted the surface being tested, such as the channel of theendoscope 500. FIGS. 3-6 illustrate a receptacle 520 suitable for use inthe present invention. It will be appreciated that receptacles used inthe embodiments of FIGS. 3-6 are typically transparent to excitation andemission wavelengths for fluorescence and to visible wavelengths oflight for visual detection.

In FIG. 3, the receptacle 520 is shown with a cap 522 having an openupper surface 524 defining an orifice 526 for allowing sampling fluidinto the vial 528. In some embodiments, the upper surface 524 comprisesa flexible material (e.g., silicone rubber) dimensioned to snugly fitover the end of a medical device (e.g., end 504 of endoscope 500) toallow sampling fluid to pass directly from the channel into the vial 528and thereby avoiding the loss of sampling fluid by splashing or thelike. Solid support member 530 is positioned within the vial 528 so thatthe area 532 treated with specific indicator is positioned to be exposedto the incoming stream of sampling fluid passing into the receptacle 520through the orifice 526 in the upper surface 524 of cap 522. The solidsupport member 530 may be positioned within the vial 528 in a frictionfit between the inner walls of the vial 528 and the edges of materialcomprising the solid support member 530. Alternatively, the solidsupport member 530 may be affixed within the vial 528 using anappropriate adhesive, for example. The specific indicator may provide acolorimetric response to the presence of bio soil or it may provide afluorescent response that can be detected through the walls of the vial528.

Referring now to FIG. 4, the construction of the receptacle is identicalto that described for FIG. 3. However, the solid support member 540 isaffixed to the inner wall of the vial 528, and specific indicator isimmobilized on strip 542 within the solid support member 540. Typically,the solid support member 540 comprises a material as described hereinwherein the solid support member 540 is affixed to the inner wall of thevial 528. Alternatively, the solid support member may comprise anadsorbent material deposited onto the inner wall of the vial 528 such assilica, cationic polymers (e.g., nylon), amine containing polymers,amine containing organosiloxanes and the like. The solid support member540 is in intimate contact with the wall of the vial 528 to minimize anycalorimetric or fluorescence absorbance by the sampling fluid that mightpartially obscure the calorimetric or fluorescent response of thespecific indicator interacting with bio soil.

In FIG. 5, the solid support member 550 comprises a strip of materialhaving an area 552 that comprises the immobilized specific indicator.The solid support member 550 is tethered to opposing sides of the wallof the vial 528 with a pair of adhesive strips 554 and 556. The solidsupport member 550 is positioned approximately centrally within the vial528 so that the specific indicator in the area 552 is also in acentralized position within the vial to facilitate contact with thesampling fluid on both sides of the solid support member 550.

FIG. 6 depicts an embodiment of the invention in which the solid supportmember is the area 560 of the inner wall of vial 528. The specificindicator is immobilized on the solid support member 560 by chemicallyimmobilizing the specific indicator on the walls of vial 528 so that acalorimetric reaction becomes visibly evident as a pattern that developson the wall. Alternatively, the specific indicator may dissolve in thesampling fluid so that the fluid becomes colored or fluorescent as areaction occurs between the specific indicator and bio soil in thesampling fluid. Typically, the vial 528 should provide a path length toenhance the limits of detection by either visual or fluorescentdetection.

Referring to FIGS. 16-18, another embodiment of the invention is shownand will now be described. Here, another form of a receptacle 600 isdepicted to capture sampling fluid drawn from a surface and into thecontainer under a vacuum pull. FIG. 16 depicts the receptacle 600 priorto allowing sampling fluid therein. A sponge 602 is positioned in thecontainer with a solid support member 604 associated with a surface ofthe sponge. Specific indicator is immobilized within the area 606 on thesolid support member 604 comprised of the materials described herein.Inlet 610 connects with the source of sampling fluid (e.g., anendoscope), and the outlet 612 allows the container to be connected to avacuum pump. In the progression depicted beginning with FIG. 16 andending at FIG. 19, the container 600 (initially empty) receives samplingfluid therein through the inlet 610 under the draw of a vacuum connectedto the outlet 612. The sponge 602 absorbs sampling fluid as it is drawninto the container 600. Depending on the volume of the sampling fluidused, the sponge 602 fills with the sampling fluid and swells, therebyfilling the container 600 with the fluid-filled sponge 602 (see FIG.18). The solid support member 604 and the specific indicator are inintimate contact with the sponge 602 so that the sampling fluid withinthe sponge 602 will contact the solid support member 604 and thespecific indicator immobilized thereon. A colorimetric or fluorescentreaction is detected on the solid support member to indicate thepresence of bio soil in the sampling fluid, as previously described.

In another embodiment, FIGS. 19-21 illustrate a container with inlet 610and outlet 612 ports, as previously described. The sponge 626, however,is substantially smaller than the sponge 602. Solid support member 604with areas 606 of immobilized specific indicator is oriented within thecontainer on top of the sponge 626. In the progression depicted in FIGS.19-21, the sponge 626 swells up upon contact with the sampling fluiduntil the filled sponge 626 blocks the openings 610 and 612 (see FIG.21). In all other respects, the embodiment of FIGS. 19-21 is the same asthat described for FIGS. 16-18.

In some embodiments, the solid support member may be positioned on orwithin a retaining member such as the receptacle discussed in theforegoing embodiments. The retaining member will typically support thesolid support member and the specific indicator with the retainingmember positioned to facilitate contact between the liquid and the solidsupport member. It will be appreciated that the retaining member may beany structure or construction that holds and retains the solid supportmember to facilitate contact between the specific indicator and theliquid after the liquid has contacted the surface being tested. In someembodiments, the retaining member is a receptacle (e.g., as aredescribed in FIGS. 3-6 and 16-21) capable of collecting and retainingthe liquid, and the solid support and specific indicator are positionedwithin the receptacle to facilitate contact between the liquid and thespecific indicator the liquid is collected in the receptacle. In someembodiments, the retaining member comprises a structure capable ofchanneling the liquid therethrough, the solid support and specificindicator positioned within the retaining member to facilitate contactbetween the liquid and the solid support member when the liquid ischanneled through the retaining member. Exemplary of structures capableof channeling the liquid therethrough include, for example, a tubularmember to having a fluid inlet through which the liquid enters theretaining member and a fluid outlet through which the liquid exits theretaining member, the solid support and specific indicator typicallypositioned within the retaining member between the fluid inlet and thefluid outlet.

As mentioned, the use of a sampling fluid with the foregoing componentsprovides a method for detecting bio soil associated with a surface,comprising:

-   -   Contacting the surface with a liquid;    -   After the liquid has contacted the surface, contacting the        liquid with the solid support member and the specific indicator        of the detector of claim 1; and    -   Inspecting the solid support member for a detectable response to        determine whether the specific indicator has interacted with        biological soil.

In this aspect of the invention, liquid is used to contact the surfacebeing sampled, to loosen and dislodge bio soil retained on the surface,and thereafter flush the dislodged soil into a vessel where the bio soilmay interact with a specific indicator. The liquid is able to probe allof the portions of the surface being sampled including the spaces withinjoints or connection areas where bio soil may become entrapped. Afterthe liquid has made contact with the surfaces being sampled, it isbrought into contact with the specific indicator immobilized on a solidsupport, as described herein. In some embodiments, the biological soildetector may utilize a liquid in a passive mode wherein the liquid isdelivered to the surface and thereafter brought into contact with thespecific indicator without assistance. In some embodiments, thebiological soil detector is provided with a vacuum to assist in thedelivery of liquid to the surface of the solid support material. Use ofliquid to loosen bio soil from all potentially contaminated surfaces ina channel of a medical device can facilitate the sampling of channelsthat are too small to be directly probed with the solid support memberaffixed to a retaining member (e.g., air and water channels of anendoscope), as described herein. Moreover, the vacuum driven embodimentsdescribed above with reference to FIGS. 16-21 should typically becompatible with hospital vacuum systems.

It will also be appreciated that the foregoing device and method of useare not limited to being used in the detection of bio soil on medicaldevices such as endoscopes. Other surfaces, such as surfaces that havecontact with food, may also be sampled to determine the presence orabsence of biological soil. Any surface may be sampled for the presenceof bio soil using the described devices and methods of the presentinvention.

Additional features of the preferred embodiments are further describedin the following non-limiting Examples.

EXAMPLES

Glossary Acronym Trade Name Generic Name Source/Address Substratechemistries (specific indicators) BCIP/NBT 3-Part Phosphatase SubstrateSystem Kirkegaard & Perry containing 5-bromo-4-chloro-3-indolylLaboratories, Inc, phosphate, p-toluidine salt, nitro blue Gaithersburg,MD tetrazolium chloride and TRIS buffer NBT/BCIP 1- Nitro bluetetrazolium chloride/5- Pierce Step ™ bromo-4-chloro-3-indolylphosphate, p- Biotechnology, Inc., Solution toluidine salt Rockford, ILX-glc or X- 5-bromo-4-chloro-3-indolyl β-D- Biosynth AG, Inc.,chemistries glucopyranoside Staad, Switzerland BCI-gal, X-gal5-bromo-4-chloro-3-indolyl β-D- Kirkegaard & Perry or X-galactopyranoside Laboratories, Inc. or chemistries Biosynth AG, Inc.BCIP, 5-bromo-4-chloro-3-indolyl Biosynth AG, Inc. X-phos or X-phosphate, p-toluidine salt phos-p-tol or X-chemistries NBT Nitro bluetetrazolium chloride TCI, Tokyo, Japan Magenta ™-5-bromo-6-chloro-3-indolyl-β-D- Biosynth AG, Inc. β-D-glcglucopyranoside Magenta ™- 5-bromo-6-chloro-3-indolyl-β-D- Biosynth AG,Inc. β-D-gal galactopyranoside Magenta ™- 5-bromo-6-chloro-3-indolylBiosynth AG, Inc. phos p-tol phosphate, p-toluidine salt5-bromo-6-chloro-3-indolyl Biosynth AG, Inc. phosphate, disodium salt4-MU-β-D-glc 4-methylumbelliferyl-β-D- Biosynth AG, Inc. glucopyranoside4-MU-β-D-gal 4-methylumbelliferyl-β-D- Biosynth AG, Inc.galactopyranoside 4-MU-phos 4-methylumbelliferyl-phosphate, p- BiosynthAG, Inc. toluidine salt esculin Sigma-Aldrich, St. Louis, MO OPAorthophthaldialdehyde Pickering Laboratories, Mountain View, CACoomassie Protein Assay Reagent Pierce Plus□ Biotechnology Inc.,Rockford, IL Enzymes Alkaline phosphatase Sigma-Aldrich, St.(Sigma-Aldrich: 4000 units/mg) Louis, MO or (Calbiochem: 35,820units/mL) Calbiochem, La (1 unit of activity = amount of enzyme Jolla,CA needed to hydrolyze 1 micromole of p- nitrophenyl phosphate/minute at25° C., pH = 9.6) β-glucosidase Sigma-Aldrich or Worthington BiochemicalCorp., Freehold, NJ β-galactosidase Worthington Biochemical Corp. orSigma-Aldrich Pseudomonas aeruginosa MBL 0484 Microbiologics, cultureSt. Cloud, MN BSA Bovine serum albumin Sigma-Aldrich Chemical Co. Calfserum HyClone, Logan, UT Solvents DMF dimethylformamide Sigma-AldrichIPA Isopropyl alcohol Sigma-Aldrich MEK Methylethylketone Sigma-AldrichSupport materials and coatings Metricel SB- Copolymer of quaternaryammonium- Pall Corporation, 6407 functional polymer and polyethersulfoneEast Hills, NY Biodyne B Cationic nylon 6,6 Pall Corporation East Hills,NY GHP-450 hydrophilic treated polypropylene Pall Corporation, membranewith 0.45 micron pore size East Hills, NY Magnaprobe Positively chargednylon membrane Osmonics, Minnetonka, MN HDPE FINATHENE High densitypolyethylene ATOFINA, 7208 Houston, TX Silica Gel Glass Backed TLCPlates (2.54 cm Whatman Inc., by 7.62 cm)(1 inch by 3 inches) Clifton,NJ EVAL or EVAL ™ Polyethylene-poly(vinyl alcohol) EVAL Company EVOHF101A copolymer of America (EVALCA), Houston, TX PP 51S07A PolypropyleneSUNOCO Chemicals, Polymer Division, Pittsburgh, PA PDAMACPoly(diallyldimethylammonium) chloride Sigma-Aldrich TIPS Thermallyinduced phase separated U.S. Pat. No. microporous membrane 4,539,256(Ex. 23) and 5,120,594 Novonette 149- Rayon/PP Nonwoven BBA Nonwovens,051 Nashville, TN 70% rayon, 30% polyester nonwoven Ahlstrom WindsorLocks LLC, Fiber Composites Division, Windsor Locks, CTω-saccharinamidoundecyltrichlorosilane U.S. Patent Application No.10/713174 (Ex. 11) (N-trimethoxysilylpropyl) United Chemicalpolyethyleneimine Technologies, Inc., Bristol, PA (Currently availablefrom Gelest, Inc. Morrisville, PA) 3-aminopropyltriethoxysilane OSiSpecialties North America, a Witco Company, South Charleston,WV(Currently available from Gelest, Inc.) N-trimethoxysilylpropyl-N,N,N-Huls America, trimethylammonium chloride Bristol, PA (Currentlyavailable from Gelest, Inc.) Buffers, solvents, additives, wettingagents Tween 80 Polyoxyethylene (20) sorbitan monooleate ICISurfactants, Wilmington, DE PBS Phosphate buffered saline, pH 7.4Examples 17, 20 TRIS tris(hydroxymethyl) amino methane Sigma-AldrichIron buffer Kirkegaard & Perry Laboratories, Inc. 1,2-propanediol J. T.Baker, a Division of Mallinckrodt, Phillipsburg, NJ glycerolSigma-Aldrich FeCl₃ Ferric Chloride Sigma-Aldrich MnCl₂ ManganeseChloride Sigma-Aldrich MgCl₂ Magnesium Chloride Sigma-Aldrich HClHydrochloric Acid J. T. Baker, a Division of Mallinckrodt GPS3-glycidoxypropyltrimethoxysilane OSi Specialties North America, a WitcoCompany (Currently available from Gelest, Inc.)

Example 1

A solution was made using BCIP/NBT 3-Part Phosphatase Substrate System(Kirkegaard & Perry Laboratories, Inc.) by combining 12.5 μL of BCIP, 50μL of NBT, 50 μL of TRIS buffer, and 37.5 μL of water. The rough side ofBiodyne B film was spotted using a micropipette to place 5 μL of thesolution made from the BCIP/NBT 3-Part Phosphatase Substrate System andair dried for 30 minutes. The spotted film was respotted with 5 μL ofalkaline phosphatase (Calbiochem) at a concentration of 3.6 units/mL.

After 26 seconds, a blue-purple spot appeared indicating the presence ofalkaline phosphatase.

Example 2

A 0.05 M TRIS buffer at pH=8.9 was prepared by mixing 50 mL 0.1 M TRISin deionized water with 7 mL 0.1N HCl and 43 mL of deionized water. Afirst solution was prepared by combining 25 mg BCIP with a mixture of 3mL 1,2-propanediol, 2 mL glycerol, and 5 mL 0.05 M TRIS buffer (pH=8.9).A second solution was prepared by combining 50 mg NBT with 3 mL1,2-propanediol, 2 mL glycerol, and 5 mL 0.05 M TRIS buffer (pH=9). Next100 μL of the first solution was mixed with 100 μL of the secondsolution, 100 μL of 0.1 mg/mL MnCl₂ in water, and 1 mL of TRIS buffer atpH=9. The resulting solution was spotted using a micropipette to place 5μL spots on the rough side Biodyne B film and the film was allowed toair dry at room temperature for 30 minutes. Next 5 μL spots of alkalinephosphatase (Calbiochem) at concentrations of 3.5 units/mL or 1.7units/mL were placed on the previously spotted film.

The average time to obtain a color response was 45 seconds for the 3.5units/mL alkaline phosphatase and 20 seconds for the 1.7 units/mLalkaline phosphatase.

Example 3

Three solutions, A, B, and C, were prepared separately. Solution A wasprepared by dissolving 25 mg of BCIP from Biosynth in 10 mL of deionizedwater. Solution B was prepared by dissolving 50 mg of NBT in 10 mL ofdeionized water. Solution C was prepared by adding 100 mg MgCl₂ and 100mg MnCl₂ to 10 mL of the TRIS buffer prepared as described in Example 2.The solutions for Runs 1-10 were prepared by combining 400 μL ofsolution A with 100 μL of solution B and 500 μL of solution C. Thesolutions for Runs 11-20 were prepared by combining 800 μL of solution Awith 100 μL of solution B and 100 μL of solution C. The solutions forRuns 21-30 were prepared by combining 300 μL of solution A with 300 μLof solution B and 400 μL of solution C. Five microliters of each of theresulting solutions were placed in spots using a micropipette on BiodyneB film and allowed to air dry for 30 minutes at room temperature. InRuns 6-10, 16-20, and 26-30, the dried films were rinsed with flowingtap water and allowed to air dry once more for 30 minutes at roomtemperature. After drying for Runs 1-30, 5 μL of alkaline phosphatase(Calbiochem) solutions with concentrations of 3.5, 1.79, 0.89, 0.45, and0.1 units/mL were each placed on the dried spots and the time needed tosee the resulting grey-black color was recorded. The results that areshown in Table 1 were averaged for three spots.

TABLE 1 Concentration of alkaline Time until color Run Numberphosphatase (units/mL) developed (seconds) 1 3.5 42 2 1.79 28 3 0.89 454 0.45 105 5 0.10 45 6 3.5 20 7 1.79 26 8 0.89 41 9 0.45 40 10 0.10 12011 3.5 49 12 1.79 96 13 0.89 57 14 0.45 96 15 0.10 120 (faint) 16 3.5 4617 1.79 39 18 0.89 50 19 0.45 99 20 0.10 154 21 3.5 20 22 1.79 29 230.89 26 24 0.45 59 25 0.10 90 26 3.5 26 27 1.79 26 28 0.89 39 29 0.45109 30 0.10 60

The color developed in under 2 minutes regardless of rinsing withflowing tap water or changing the concentration of the BCIP. Theconcentration of the enzyme reached a limit of detection for Run 20.

Example 4

A 1× concentration of 50 μL BCI-gal/2 mL of iron buffer solution wasprepared according to the manufacturer's instructions (Kirkegaard &Perry Laboratories, Inc.). A 2× concentration of 100 μL BCI-gal/2 mL ofiron buffer solution was prepared. Next a 4× concentration of 100 μLBCI-gal/1 mL of iron buffer solution was prepared. Each of thesolutions, 1×, 2×, and 4× were spotted on an Osmonics positively chargednylon membrane using a micropipette to place 10 μL drops and the filmwas allowed to air dry at room temperature for 30 minutes. Next 10 μLdrops of β-galactosidase (supplied at 590 units/mL by (Sigma-Aldrich) inconcentrations of 5.9, 0.59, 0.059, and 0.0059 units/spot or 0.5, 0.05,0.005, 0.0005 mg/spot. The time needed for development of color wasrecorded in seconds. The results are shown in Table 2.

TABLE 2 Time to color development for BCI-gal/iron β-galactosidasebuffer concentration Run concentration 1× 2× 4× Number (mg/spot)(units/spot) (seconds) (seconds) (seconds) 1 0.5 5.9 10 10  7 2 0.050.59 30 30 17 3 0.005 0.059 43 37 25 4 0.0005 0.0059 >120 >120 120(faint)

Example 5

A solution of esculin (1 mg/mL)/FeCl₃ (1 mg/mL) was spotted on the roughside of Biodyne B film using a micropipette to place 5 μL drops and thefilm was allowed to air dry at room temperature for 30 minutes. Next 5μL drops of β-glucosidase concentrations of 625, 312.5, 156.25, 78.125,and 39.0625 were placed on the esculin/FeCl₃ spots and the time neededfor development of color was recorded. esculin worked well with pureenzyme systems. The results are shown in Table 3.

TABLE 3 β-glucosidase Time for development of Run (units/mL) color(seconds) 1 625.0000 5 2 312.5000 5 3 156.2500 10 4 78.1250 20 5 39.062520

Example 6

Test tubes were filled with 100 μL a solution of esculin (1 mg/mL)/FeCl₃(1 mg/mL). Next 100 μL of β-glucosidase at concentrations of 5, 0.5,0.25, 0.125, 0.0625, 0.05, 0.025, and 0.0025 units/mL were dropped intothe test tubes containing the esculin/FeCl₃ solutions and the timenecessary for development of color change from green to black wasrecorded. The results are shown in Table 4.

TABLE 4 β-glucosidase Time for development of Run (units/mL) color(seconds) 1 5 instant 2 0.5 10 3 0.25 30 4 0.125 60 5 0.0625 120 6 0.05150 7 0.025 300 8 0.0025 600 Control Pure water No change

Example 7

A solution of esculin (1 mg/mL)/FeCl₃ (1 mg/mL) was spotted on the roughside of Biodyne B film using a micropipette to place 5 μL drops and thefilm was allowed to air dry at room temperature for 30 minutes. Clinicalendoscope soil samples (patient soil) were collected by flushing 10 mLof phosphate buffered saline through the biopsy lumen of a colon scopefollowing a colonoscopy procedure at Mayo Clinic in Rochester, Minn.Then 100 μL of the clinical endoscope soil samples was placed on theesculin/FeCl₃ spots and the time necessary for development of color wasrecorded. Color development took longer than 10 minutes.

Example 8

Four samples of a Metricel SB-6407 membrane were spotted using amicropipette to place 10 μL of BCIP/NBT solution per spot. The spotswere allowed to air dry at room temperature. One spotted membrane wasrespotted with 10 μL alkaline phosphatase (Sigma-Aldrich) solution (500μg/mL alkaline phosphatase in distilled water) per spot. Two of theBCIP/NBT spotted membranes were respotted with 10 μL (per spot) of fluidsamples obtained from a used gastrointestinal endoscope prior to manualcleaning and after manual cleaning respectively. The fourth BCIP/NBTspotted membrane was respotted with 10 μL distilled water per spot.

A blue-purple color due to product of the reaction of enzyme andindicator appeared within 2 minutes of addition of enzyme in both thepure sample and the pre-cleaning sample. The post-cleaning sample didnot exhibit a visible calorimetric response within 2 minutes, nor didthe spots treated with distilled water alone. The results are shown inTable 5.

TABLE 5 Color Change within 2 minutes of addition of Run Treatmentenzyme 1 Alkaline phosphatase solution Colorless to blue-purple color 2Samples from used Colorless to blue-purple color gastrointestinalendoscope prior to manual cleaning 3 Samples from used No color changegastrointestinal endoscope after manual cleaning 4 Distilled water Nocolor change

Example 9

A HDPE TIPS membrane was made according to the process described in U.S.Pat. No. 4,539,256 (Example 23) except instead of extruding into a waterquench bath, the extruded membrane was taken onto a chilled patternedcasting wheel as described in U.S. Pat. No. 5,120,594.

Several HDPE TIPS membranes were fastened to a metal frame, coated bypouring a 2.5% EVOH in 60:40 IPA:water solution and spreading using arubber spreader to smooth and remove excess. Coated membranes wereallowed to dry at room temperature overnight. Six samples were coated bypouring 5:1, 2:1 and 1:1 solutions of poly(diallyldimethylammonium)chloride (PDAMAC) (<100,000 MW supplied in 40 wt % water) in deionizedwater and 5:1, 2:1 and 1:1 solutions of poly(diallyldimethylammonium)chloride (100,000-200,000 MW supplied in 20 wt % water) in deionizedwater and allowed to air dry at ambient temperature. The EVOH- andPDAMAC-coated membranes were spotted with 10 μL BCIP/NBT solution perspot as in Example 1 and allowed to dry. These spots then were respottedwith 10 μL alkaline phosphatase (Sigma-Aldrich) solution per spot at aconcentration of 500 μg/mL in distilled water.

A blue-purple calorimetric response was visible within 2 minutes on thePDAMAC-coated samples, but not on the samples coated with EVOH alone. Onboth of the PDAMAC-coated samples, the rate of the response was5:1>2:1>1:1. The results are shown in Table 6.

TABLE 6 Color Time Run Coating Treatment Change (minutes) 1 2.5% EVOH in60 Alkaline No change 2 IPA:40 water phosphatase 2 5:1 EVOH:PDAMACAlkaline Colorless to 2 (<100K MW) phosphatase blue-purple 3 2:1EVOH:PDAMAC Alkaline Colorless to 2 (<100K MW) phosphatase blue-purple 41:1 EVOH:PDAMAC Alkaline Colorless to 2 (<100K MW) phosphataseblue-purple 5 5:1 EVOH:PDAMAC Alkaline Colorless to 2 (100-200K MW)phosphatase blue-purple 6 2:1 EVOH:PDAMAC Alkaline Colorless to 2(100-200K MW) phosphatase blue-purple 7 1:1 EVOH:PDAMAC AlkalineColorless to 2 (100-200K MW) phosphatase blue-purple

Example 10

Using a micropipette approximately 5 microliters of BCIP/NBT solutionper spot was placed onto Silica Gel Glass Backed TLC Plates (2.54cm×7.62 cm). The spots were dried using warm air supplied by a Model#HG-751 heat gun (Master Appliance Corp., Racine, Wis.). One of thespotted glass plates was used as a control and was not placed in a waterbath. The other spotted glass plates (test plates) were placed into awater bath (25° C.) for 2 minutes, removed, and dried again using theheat gun. Next 5 microliters of alkaline phosphatase (Sigma-Aldrich)solution (25 mg phosphatase/mL water) was placed on the indicator spotson the test plates and the control plate. For all enzyme indicatorcombinations, a purple color due to reacted indicator was noticeablewithin two minutes. Subsequently, for all colored spots on both controlplate and test plates, the color was not washed away when they wereplaced into a water bath (25° C.). The results of this experimentindicated that spotted enzyme indicator remained bound to silica evenafter being washed with water and that it reacted with enzyme in itsbound state. Additionally, the experiment demonstrated that reactedenzyme indicator remains bound to silica in the presence of water.

Example 11

Metricel SB-6407 membrane (comprising quaternary ammonium groups,available from Pall) was spotted as in Example 1 with BCIP/NBT, rinsedwith flowing tap water, dried, and allowed to react with alkalinephosphatase (Sigma-Aldrich). A blue-purple color due to product of thereaction of enzyme and indicator appeared within 2 minutes of additionof enzyme and could not be washed away with water.

Comparative Example A

A TexWipe swab (Item # TX712A from Texwipe Co, Inc., Upper Saddle River,N.J.) was treated in the same manner as the Metricel membrane in Example11. When the swab was washed with water as in Example 11, the indicatorwas washed away from the swab. Additionally, color resulting fromreaction of indicator and enzyme on the TexWipe swabs was readily washedaway when rinsed with flowing tap water.

Comparative Example B

A support consisting of an uncoated PP TIPS membrane was prepared by thefollowing steps: securing the membrane in a hoop to avoid shrinkageduring the drying step; loading membrane with 2 mL of X-glc solution ata concentration of 0.0003 g/mL in DMF; and drying the membrane for 20min at 58° C. The membrane was then tested for a calorimetric responseby placing spots of 10 μl pure β-glucosidase solution at a concentration100 units/mL on the loaded membrane using a micropipette and recordingthe time necessary for development of color. The aqueous enzyme solutiondid not wet the membrane when it was spotted onto the surface. Noresponse was observed.

Example 12

A HDPE TIPS membrane was coated by dispensing and evenly spreadingapproximately 1 mL of EVAL solution (2.8% (w/w) EVAL in 60:40 isopropylalcohol:water) using a plastic pipette. The coated membrane was furtherprepared by the following steps: securing the membrane in a 10.08 cm indiameter hoop to avoid shrinkage during the drying step; loadingmembrane with 2 mL of X-glc solution at a concentration of 0.15 g/mL;and drying the membrane for 20 min at 58° C. in an incubator (PrecisionMechanical Convection Incubator from GCA Corporation, Andover, Mass.).

The membrane was then tested at room temperature for colorimetricresponse by using a micropipette to place 10 μL of β-glucosidasesolution in spots at concentrations of 100, 50, 25, 12.5, 6.3 and 3.1units/mL in reagent-grade water on the loaded membrane and recording thetime necessary for development of the color. In Run 6, when the colordid not develop after 50 minutes at room temperature, the membraneincluding Runs 1-6 was heated to 58° C. in an incubator for 10 minutes.Then the membrane was removed from the incubator and allowed to cool toroom temperature. The color continued to develop in the examplesovernight at room temperature. Results are shown in Table 7.

TABLE 7 β-glucosidase dilutions Run Number (units/mL) Time (minutes) 1100  1.5 2 50  2 3 25  2.5 4 12.5  5.0 5 6.3  6.0 6 3.1 10¹ ¹After 50minutes at room temperature and 10 minutes at 58° C. in an incubator.

Example 13

A rayon/PP nonwoven support was prepared by the following steps:securing the nonwoven in a hoop to avoid shrinkage during the dryingstep; loading the nonwoven support with 2.5 mL of X-glc solution at aconcentration of 0.15 g/mL in DMF; and drying the membrane for 20 min at58° C. The X-glc loaded nonwoven support was then tested at roomtemperature for calorimetric response by using a micropipette to place10 μL of β-glucosidase solution in spots at concentrations of 100, 50,25, 12.5, 6.3 and 3.1 units/mL in reagent-grade water on the loadedsupport and recording the time necessary for development of the color.In Run 6, when the color did not develop after 21 minutes at roomtemperature, the membrane including Runs 1-6 was heated to 58° C. in anincubator for 10 minutes. Then the membrane was removed from theincubator and allowed to cool to room temperature. Color continued todevelop overnight. The contrast between the colorimetric response andthe textured background made the detection of color more obvious soonerthan on the TIPS membrane support in Example 12. Results are shown inTable 8.

TABLE 8 β-glucosidase dilutions Run Number (units/mL) Time (minutes) 1100  1.0 2 50  1.5 3 25  2.0 4 12.5  3.0 5 6.3  4.0 6 3.1 10¹ ¹After 21minutes at room temperature and 10 minutes at 58° C. in an incubator.

Example 14

A HDPE TIPS membrane support was prepared by the following steps:securing the membrane in a hoop to avoid shrinkage during the dryingstep; spotting the membrane with EVAL solution at 2.8% (w/w) EVAL in60:40 isopropyl alcohol: water using a plastic pipette (SAMCO TransferPipettes, San Fernando, Calif.); air drying the spotted membraneovernight at 25° C.; loading the spots on membrane with 2 mL of X-glcchemistry at a concentration of 0.22 g/mL in DMF; and drying the spottedmembrane for 20 min at 58° C. The membrane was wettable by the aqueousenzyme solutions only where EVAL was spotted onto the surface.

The membrane was then tested for colorimetric response by using amicropipette to place 10 μL of β-glucosidase solution onto theEVAL/X-glc spots at concentrations of 100, 50, 25, 12.5, 6.3 and 3.1units/mL in reagent-grade water on the loaded membrane and recording thetime necessary for development of color. Color continued to developovernight. The colors were more vibrant than with the EVAL-coated HDPE(Example 12) because the sample was confined to a specific area, but theresponse did not develop any faster. Results are shown in Table 9.

TABLE 9 β-glucosidase dilutions Run Number (units/mL) Time (minutes) 1100 1.5 2 50 3.0 3 25 4.0 4 12.5 5.5 5 6.3 7.0 6 3.1 12.0

Example 15

A HDPE TIPS membrane support was prepared by the following steps:securing the membrane in a hoop to avoid shrinkage during the dryingstep; spotting the membrane with EVAL solution at 2.8% (w/w) EVAL in60:40 isopropyl alcohol: water using a plastic pipette; drying thespotted membrane overnight at 25° C.; loading some of the hydrophilicspots on membrane with 25 μL of X-glc at a concentration of 0.15 g/mL inDMF or with 25 μL of X-gal (Biosynth AG, Inc.) at a concentration of0.15 g/mL in DMF; drying the spotted membrane for 5 min at 58° C.;loading other hydrophilic spots on the membrane with 25 μL of CoomassiePlus™ Protein Assay Reagent; and drying for 20 minutes at 58° C.

The spotted membrane was then tested for calorimetric response by usinga micropipette to place 20 μL of β-glucosidase (Sigma-Aldrich) inreagent-grade water or 20 μL of β-galactosidase (Worthington BiochemicalCorp.) in reagent-grade water onto the EVAL/X-glc or EVAL/X-gal spots,respectively, with a concentration of 100 units/mL, and recording thetime necessary for development of color.

Additionally Pseudomonas aeruginosa MBL 0484 from Microbiologics wasgrown overnight (16 hours) at 37° C. in tryptic soy broth (BectonDickinson and Company, Sparks, Md.) and was used to test the spottedmembrane for a calorimetric response. A micropipette was used to place20 μL of serial 1:10 dilutions of Pseudomonas aeruginosa (10⁸ CFU/mL) incalf serum on each type of indicator-loaded hydrophilic spot.

The enzyme, β-glucosidase in reagent-grade water, produced a responsewithin 1.5 minutes for the X-glc chemistry. While the enzyme,β-galactosidase in reagent-grade water produced a blue response within 1minute for the X-gal chemistry. The Coomassie chemistry produced aresponse within 20 seconds for every dilution of Pseudomonas aeruginosaculture. The 10⁸ CFU/mL Pseudomonas aeruginosa culture did not produce aresponse for the X-gal or X-glc chemistries.

Example 16

A HDPE TIPS membrane support was prepared by the following steps:securing the membrane in a hoop to avoid shrinkage during the dryingstep; spotting the membrane with EVAL solution at 2.8% (w/w) EVAL in60:40 isopropyl alcohol: water using a plastic pipette; drying thespotted membrane overnight at 25° C.; loading six hydrophilic spots onmembrane with 20 μL of a combination of X-glc, X-gal (Biosynth AG,Inc.), and X-phos-p-tol chemistries at a concentration of 0.07 g/mL ofeach in DMF; loading another six hydrophilic spots on the membrane with20 μl of a combination of Magenta™-glc, Magenta™-gal, andMagenta™-phos-p-tol chemistries at a concentration of 0.06 g/mL of eachin DMF; and drying for 20 minutes at 58° C.

The membrane was then tested for calorimetric response by using amicropipette to place 20 μL of β-glucosidase (Sigma-Aldrich) inreagent-grade water (100 units/mL), 20 μL of β-galactosidase(Worthington Biochemical Corp.) in reagent-grade water (100 units/mL),and 20 μL of calf serum, 20 μL clinical endoscope soil samples (MayoClinic, Rochester, Minn.) for each type of coated hydrophilic spot onthe loaded membrane and recording the time necessary for development ofcolor. Clinical endoscope soil samples (patient soil) were collected byflushing 10 mL of phosphate buffered saline through the biopsy lumen ofa colonscope following colonoscopy procedure and following the cleaningprocedure for the scope.

The β-glucosidase reacted within 1 minute on the Magenta™ combinationand within 2 minutes on the X-chemistries combination. β-galactosidasereacted within 2 minutes for each of the X-chemistry combinations andMagenta™ chemistry combinations. The calf serum did not react within 10minutes. The PB78 clinical sample from Mayo reacted within 4 minutes forthe Magenta™-glc, Magenta™-gal, or Magenta™-phos-p-tol chemistrycombination and within 6 minutes for the X-glc, X-gal, or X-phos-p-tolchemistry combination.

Example 17

A HDPE TIPS membrane support was placed in a hoop, spotted with EVALsolution and allowed to dry as described in Example 15. The spottedmembrane was subsequently coated with a 1.5 mL of 1:1000 ofco-saccharinamidoundecyltrichlorosilane in hexadecane, allowed to reactfor 20 minutes, washed with MEK and allowed to air dry three times.After drying, the hydrophilic spots on the treated membrane were spottedwith 20 μL of a combination of X-glc, X-gal (Biosynth AG, Inc.), andX-phos-p-tol chemistries in DMF at a concentration of 0.2 g totalsubstrate/mL (0.07 g X-chemistry/mL for each chemistry) and allowed toreact for approximately 20 minutes. A PBS Buffer, pH 7.4 was prepared bycombining 0.14 M NaCl (EM Science, Gibbstown, N.J.) 0.006 M K₂HPO₄(Sigma-Aldrich) and 0.02 M KH₂PO₄ (Sigma-Aldrich). The treated membranewas washed twice with the PBS Buffer and with 1% Tween™ 80 and allowedto air dry.

The hydrophilic spots on the membrane were then tested for colorimetricresponse with 20 μL of β-glucosidase (Sigma-Aldrich), β-galactosidase(Worthington Biochemical Corp.), calf serum, and clinical endoscope soilsamples prepared as in Example 16 and the time needed for development ofcolor was recorded. The time needed to develop a color was not within a2-minute time interval, but color did develop within two hours forβ-glucosidase, β-galactosidase, and one soiled endoscope sample with thecombination of X-gal, X-glc, and X-phos.

Example 18

A HDPE TIPS membrane support was prepared by securing the membrane inhoop to avoid shrinkage during the drying step, coating the membranewith EVAL as described in Example 12, loading the coated membrane with 2mL of 4-MU-β-D-glc chemistry at a concentration of 0.0003 g/mL, anddrying for 20 min at 58° C.

The membrane was then tested for fluorescent response by spotting theloaded membrane with 10 μL of pure β-glucosidase (Sigma-Aldrich)solution and at 50, 25, 12.5, 6.3, and 3.1 units/mL dilutions in reagentgrade water, exposing the membrane to UV light (365 nm) until afluorescent response developed and recording the time. The results areshown in Table 10.

TABLE 10 β-glucosidase dilutions Run Number (units/mL) Time (seconds) 1100 instant 2 50 20 3 25 30 4 12.5 30 5 6.3 45 6 3.1 85

Example 19

A rayon/PP nonwoven support was prepared by securing the nonwoven in ahoop to avoid shrinkage during the drying step, coating the nonwovenwith 3 mL of 4-MU-β-D-glc chemistry at a concentration of 0.0003 g/mL inDMF, and drying for 20 min at 58° C. The membrane was then tested forfluorescent response by spotting the loaded membrane with 10 μL of pureβ-glucosidase (Sigma-Aldrich) solution and at 50, 25, 12.5, 6.3, and 3.1units/mL dilutions in reagent grade water, exposing the membrane to UVlight (365 nm) until a fluorescent response developed and recording thetime. The results are shown in Table 11.

TABLE 11 Run β-glucosidase dilutions Time Number (units/mL) (seconds) 1100 instant 2 50 20 3 25 30 4 12.5 30 5 6.3 60 6 3.1 90

Example 20

A HDPE TIPS membrane support was placed in a hoop, coated with EVALsolution as described in Example 18 subsequently coated with 1.5 mL of1:1000 ω-saccharinamidoundecyltrichlorosilane in hexadecane, allowed toreact for 20 minutes, washed with MEK and allowed to air dry threetimes. After drying, the treated membrane was coated with 20 μL of asolution of 4-MU-β-D-glc chemistry at a concentration of 0.0003 g/mL inDMF, washed twice with the PBS Buffer, pH 7.4, prepared as described inExample 17 and with 1% Tween™ 80 and allowed to air dry.

The coated membrane was then tested for fluorescent response by placing10 μL of β-glucosidase (Sigma-Aldrich) in spots using a micropipette onthe membrane, exposing the membrane to UV light (365 nm) until afluorescent response developed and recording the time. The fluorescenceresponse for enzyme solution at 100 units/mL was instantaneous.

Example 21

A GHP-450 membrane support was prepared by securing the membrane in ahoop to avoid shrinkage during the drying step, coating the membranewith 3 mL of 4-MU-β-D-glc chemistry at a concentration of 0.0003 g/mL inDMF, and drying for 20 min at 58° C. The membrane was then tested forfluorescent response by spotting the loaded membrane with 10 μL of pureβ-glucosidase (Sigma-Aldrich) solution and at 50, 25, 12.5, 6.3, and 3.1units/mL dilutions in reagent grade water, exposing the membrane to UVlight (365 nm) until a fluorescent response developed and recording thetime. The results are shown in Table 12.

TABLE 12 Run β-glucosidase dilutions Time Number (units/mL) (seconds) 1100 instant 2 50 instant 3 25 instant 4 12.5 60 5 6.3 90 6 3.1 120 

Example 22

A HDPE TIPS membrane support was prepared by securing the membrane in ahoop to avoid shrinkage during the drying step, coating the membranewith EVAL as described in Example 12, subsequently coating with 2 mL ofOPA solution at a concentration of 0.8 mg/mL, and drying for 30 min at58° C. The membrane was then tested for fluorescent response by spottingthe loaded membrane with 10 μL of BSA solution at 2, 1, 0.5, 0.25, 0.13and 0.06 mg/mL in reagent grade water, exposing the membrane to UV light(365 nm) until a fluorescent response developed and recording the time.The results are shown in Table 13.

TABLE 13 Run BSA dilutions Time Number (mg/mL) (minutes) 1 2  1 2 1  1.5 3 0.5   3.5 4 0.25  5 5 0.13 >10¹ 6 0.06 >10¹ ¹No response wasobserved within 10 minutes.

Example 23

A rayon/PP nonwoven support was prepared by securing the nonwoven in ahoop to avoid shrinkage during the drying step, loading the membranewith 3 mL of OPA solution at a concentration of 0.8 mg/mL, and dryingfor 30 min at 58° C. The nonwoven support was then tested forfluorescent response by spotting the loaded membrane using amicropipette with 10 μL of BSA solution at 2, 1, 0.5, 0.25, 0.13 and0.06 mg/mL in reagent-grade water, exposing the membrane to UV light(365 nm) until a fluorescent response developed and recording the time.The results are shown in Table 14.

TABLE 14 Run BSA dilutions Time Number (mg/mL) (minutes) 1 2  1 2 1  2.5 3 0.5 >10¹ 4 0.25 >10¹ 5 0.13 >10¹ 6 0.06 >10¹ ¹No response wasobserved within 10 minutes.

Example 24

Two solutions were prepared by first dissolving 25 mg of5-bromo-6-chloro-3-indolyl phosphate, disodium salt in 10 mL ofdeionized water and then dissolving 50 mg of NBT was dissolved in 10 mLof deionized water. Next four 10 mL TRIS buffer solutions (A, B, C, D)were prepared as described in Example 2. To Buffer Solution A, 250 mg ofMnCl₂ was added. To Buffer solution B, 250 mg of MgCl₂ was added. ToBuffer solution C, 250 mg of MgCl₂ and 250 mg of MnCl₂ were added.Buffer solution D had no metal salt additions. Four5-bromo-6-chloro-3-indolyl phosphate, disodium salt/NBT/Buffer solutionswere prepared by combining 400 μL of 5-bromo-6-chloro-3-indolylphosphate, disodium salt/water solution, 100 μL NBT/water solution, and500 μL of Buffer solution A, B, C, or D. The resulting four indicatorsolutions were labeled indicator solution 1, 2, 3, and 4 respectively.Five microliters of each of the four solutions was placed in spots on aBiodyne B film using a micropipette. The spots were allowed to air dryfor 30 minutes at room temperature. Each spot was respotted with 5microliters of alkaline phosphatase (Calbiochem) at a concentration of1.79 units/mL. The time needed to develop color, the initial indicatorspot color on the film, and the color of the indicator spot after ageingat ambient temperature and light conditions for 1 day and for 1 weekwere recorded.

The samples containing MnCl₂ and MgCl₂ showed shortened times for colordevelopment with the alkaline phosphatase enzyme. Samples with MgCl₂alone resulted in a spotty color that aged in the same manner asIndicator Solution 4 (containing neither MnCl₂ nor MgCl₂). The yellowcolor of manganese salt-containing spots gave a good contrast betweenthe unreacted indicator and that of the brown-black color of the reactedindicator. Results are shown in Table 15.

TABLE 15 Indicator Initial Indicator Indicator Solution Time to developindicator spot color spot color Number color (seconds) spot color after1 day after 1 week 1 40 Yellow Yellow Yellow 2 17 Colorless PurplePurple 3 30 Yellow Yellow Yellow 4 110 Colorless Purple Purple

Example 25

A HDPE TIPS membrane support was placed in a hoop, spotted with EVALsolution and allowed to dry as described in Example 15. Then eachhydrophilic spot on the membrane was loaded with 10 μL of X-gal solutionprepared by combining 200 μL of X-gal (Kirkegaard & Perry Laboratories,Inc.) with 1 mL iron buffer; 10 μL BCIP/NBT (3-Part PhosphataseSubstrate System from Kirkegaard & Perry Laboratories, Inc.) prepared bycombining 4 mL of BCIP, 1 mL of NBT, and 5 mL of TRIS buffer; 10 μL of4-MU-β-D-gal prepared by combining 0.3 g/L of water and autoclaving for15 minutes at 121° C.; 10 μL of 4-MU-P-D-glc prepared by combining 0.3g/L of water and autoclaving for 15 minutes at 121° C.; or 10 μL of4-MU-phos prepared by dissolving 0.03 g in 100 microliters DMF anddiluting with 100 mL of water. The solutions were placed on the membraneusing a micropipette and allowed to air dry at room temperature.

The membrane was then tested for colorimetric response by using amicropipette to place 10 μL of β-galactosidase (Sigma) in reagent-gradewater at concentrations of 188, 18.8, 9.4, and 1.88 units/mL, 10 μL ofβ-glucosidase (Worthington Biochemical Corp.) in reagent-grade water atconcentrations 50, 5, 2.5 an 0.5 units/mL, or 10 μL alkaline phosphatase(Calbiochem) in reagent-grade water at concentrations of 3582, 358,35.8, 3.58, 0.358 units/mL for each type of coated hydrophilic spot onthe loaded membrane and recording the time necessary for development ofcolor. The results are shown in Table 16.

TABLE 16 Enzyme Time to concen- develop Run Indicator tration colorNumber Chemistry Enzyme tested (units/mL) (seconds)  1 BCIP/NBT alkaline358 10 phosphatase  2 BCIP/NBT alkaline 35.8 20 phosphatase  3 BCIP/NBTalkaline 3.58 30 phosphatase  4 BCIP/NBT alkaline 0.358 90 phosphataseControl A BCIP/NBT water 0 No Change  5 X-gal β-galactosidase 188 45  6X-gal β-galactosidase 18.8 60  7 X-gal β-galactosidase 9.4 105  8 X-galβ-galactosidase 1.88 180  9 MU-gal β-galactosidase 188 instant 10 MU-galβ-galactosidase 18.8 10 11 MU-gal β-galactosidase 9.4 15 12 MU-galβ-galactosidase 1.88 30 13 4-MU-β-D-glc β-galactosidase 50 instant 144-MU-β-D-glc β-galactosidase 5 5 15 4-MU-β-D-glc β-galactosidase 2.5 516 4-MU-β-D-glc β-galactosidase 0.5 10 17 MU-phos alkaline 3582 instantphosphatase 18 MU-phos alkaline 358 10 phosphatase 19 MU-phos alkaline35.8 15 phosphatase 20 MU-phos alkaline 3.58 60 phosphatase 21 MU-phosalkaline 0.358 105 phosphatase Control B MU-phos water 0 No Change

Example 26

A HDPE TIPS membrane support was prepared by securing the membrane in ahoop to avoid shrinkage during the drying step, coating the membranewith EVAL as described in Example 12, subsequently placing 6 drops of 10μL BCIP/NBT (3-Part Phosphatase Substrate System from Kirkegaard & PerryLaboratories, Inc.) prepared by combining 4 mL of BCIP, 1 mL of NBT, and5 mL of TRIS buffer in spots using a micro pipette and allowing to airdry at room temperature.

The membrane was then tested for colorimetric response by using amicropipette to place 10 μL of alkaline phosphatase (Calbiochem) inreagent-grade water at concentrations of 3582, 358, 35.8, 3.58, 0.358units/mL on the 5 spots of BCIP/NBT. The sixth BCIP/NBT spot was testedin the same manner with 10 μL of water. The time necessary fordevelopment of color was recorded. The results are shown in Table 17

TABLE 17 Enzyme concentration Time to develop Run Number Enzyme tested(units/mL) color (seconds) 1 alkaline phosphatase 3582  5 2 alkalinephosphatase 358 15 3 alkaline phosphatase 35.8 25 4 alkaline phosphatase3.58 30 5 alkaline phosphatase 0.358 70 6 Water (no enzyme) 0 No Change

Example 27

By first dissolving 5 mg of 5-bromo-6-chloro-3-indolyl phosphate,disodium salt in 10 mL deionized water and then dissolving 50 mg of NBTin 10 mL deionized water, two solutions were prepared. Next a 10 mL TRISbuffer solution was prepared as described in Example 2 and 100 mg ofMnCl₂ and 100 mg of MgCl2 were added. Then, 300 microliters of the5-bromo-6-chloro-3-indolyl phosphate solution and 300 microliters of theNBT solution were combined with 400 microliters of the TRIS buffersolution containing MgCl₂ and MnCl₂. Five microliters of the indicatorsolution was used to place thirty spots (1 mm diameter and 1 mm apart)onto a piece of a Biodyne B film (1 cm×3 cm) using a micropipette. Apolyethylene tube simulating the inside of the biopsy channel of anendoscope was filled with 2 mL of contents flushed from a patient soiledendoscope and then emptied. Within 10 seconds the spotted Biodyne B filmwas pushed through the lumen of the soiled polyethylene tube. Acolorimetric reaction occurred within 20 seconds, thus indicating thepresence of alkaline phosphatase in the polyethylene lumen that had beensoiled with the patient soiled endoscope contents.

Example 28

Several 70% rayon/30% polyester nonwoven supports were treated withthree amine-containing silanes: (N-trimethoxysilylpropyl)polyethyleneimine, 3-aminopropyltriethoxysilane, andN-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride; and a wettingagent, 3-glycidoxypropyltrimethoxysilane (GPS) by dip coating. First aslightly acidic solution of 95 percent water/5 percent ethanol wasprepared by adding sulfuric acid drop wise to obtain pH=4. GPS and eachaminosilane were added to the acidic solution in a ratio May 5, 1990,respectively. After the nonwoven was dipped in one of the aminosilanesolutions, it was dipped into two sequential ethanol baths and heatcured at 70° C. for 1½ to 2 hours in an oven (Commercially available asModel LFD1-42-3 from Despatch, Lakeville, Minn.). For comparison to theamine-containing nonwovens, one sample of the nonwoven was leftuntreated and one sample was only treated with GPS (no amine groupspresent).

A micropipette was used to place several 5-microliter spots of NBT/BCIP1-Step™ Solution onto each of the three aminosilane-treated samples, theuntreated sample and the GPS-treated sample. These spots were allowed toair dry at room temperature for at least 30 minutes. Next, 5 microlitersof alkaline phosphatase (Calbiochem) at concentrations of 3.5, 1.75,0.35, and 0.175 units/mL were placed onto the dried spots of NBT/BCIP onthe nonwoven support using a micropipette. For comparison, 5 microlitersof sterile, ultrapure water (0 units/mL of the enzyme) were also placedon additional dried spots of NBT/BCIP using a micropipette. The timeneeded for the first appearance of color was recorded and the resultsare shown in Table 18.

To determine if the background color of the unreacted NBT/BCIP 1-Step™Solution changed over time, some of the unreacted dried spots ofNBT/BCIP on the nonwoven support were observed for the initial color andthe color change after 1 day. These observations are shown in Table 19.

TABLE 18 Enzyme- Time to alkaline develop Run phosphatase color NumberTreated with Aminosilanes and GPS (units/mL) (seconds) 1 None(Untreated) 0 (Water No only) Change¹ 2 None (Untreated) 0.175 150  3None (Untreated) 0.350 90 4 None (Untreated) 1.75 55 5 None (Untreated)3.50 30 6 (N-trimethoxysilylpropyl) 0 (Water No polyethyleneimine only)Change¹ 7 (N-trimethoxysilylpropyl) 0.175 45 polyethyleneimine 8(N-trimethoxysilylpropyl) 0.350 41 polyethyleneimine 9(N-trimethoxysilylpropyl) 1.75  30² polyethyleneimine 10(N-trimethoxysilylpropyl) 3.50  20² polyethyleneimine 113-aminopropyltriethoxysilane 0 (Water No only) Change¹ 123-aminopropyltriethoxysilane 1.75   6³ 13 3-aminopropyltriethoxysilane3.50   7³ 14 N-trimethoxysilylpropyl-N,N,N- 0 (Water Notrimethylammonium chloride only) Change¹ 15N-trimethoxysilylpropyl-N,N,N- 0.175 35 trimethylammonium chloride 16N-trimethoxysilylpropyl-N,N,N- 0.350 25 trimethylammonium chloride 17N-trimethoxysilylpropyl-N,N,N- 1.75  13³ trimethylammonium chloride 18N-trimethoxysilylpropyl-N,N,N- 3.50  13³ trimethylammonium chloride 19GPS only 0 (Water No only) Change¹ 20 GPS only 1.75  8 21 GPS only 3.50 9 ¹No change within 10 minutes ²Colors darken overnight ³Colors stableafter 1 week.

TABLE 19 Color of Observation Initial Indicator Run Treated withIndicator Spot Number Aminosilanes and GPS Spot Color after 1 day 22Untreated Colorless Faint purple 23 N-trimethoxysilylpropyl-N,N,N-Colorless Faint purple trimethylammonium chloride 24(N-trimethoxysilylpropyl) Colorless Purple polyethyleneimine

Example 29

Several 70% rayon/30% polyester nonwoven supports were treated with oneof three amine-containing silanes: (N-trimethoxysilylpropyl)polyethyleneimine, 3-aminopropyltriethoxysilane, andN-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride; and a wettingagent, 3-glycidoxypropyltrimethoxysilane (GPS) as described for Example28. Additionally, one sample of the nonwoven was left untreated. Threesolutions, labeled solution A, solution B, and solution C, were preparedin a similar manner to the solutions described in Examples 3 and 24.Solution A was prepared by combining 25 mg of BCIP with 10 mL deionizedwater. Solution B was prepared by dissolving 15 mg NBT in 10 mLdeionized water. Solution C was prepared by combining 250 mg each MnCl₂and MgCl₂ and dissolving in 10 mL of TRIS buffer (pH=8.9). The TRISbuffer was prepared according to the description for Example 2. Theindicator solution was prepared by combining 400 microliters of solutionA, 100 microliters of solution B, and 500 microliters of solution C,yielding a ratio of 4:1:5 BCIP:NBT:TRIS buffer with MnCl₂ and MgCl₂salts. A micropipette was used to place several 5-microliter drops ofthe 4:1:5 indicator solution in a spotted pattern onto each of the threeaminosilane treated samples and an untreated sample. These spots wereallowed to air dry at room temperature for at least 30 minutes. Next, 5microliters of alkaline phosphatase (Calbiochem) at concentrations of1.00, 0.50, 0.10, 0.05, and 0.01 units/mL were placed onto the driedspots of indicator solution using a micropipette. For comparison, 5microliters of deionized water (0 units/mL of the enzyme) were alsoplaced on additional dried spots of indicator solution using amicropipette. The time needed for the first appearance of thecharacteristic purple-black color was recorded and the results are shownin Table 20.

To determine if the background color of the unreacted 4:1:5BCIP:NBT:TRIS buffer and MnCl₂ and MgCl₂ salts changed over time, someof the unreacted dried spots on the nonwoven support were observed forthe initial color and the color change after 1 day (dry). Additionallyto determine the stability of the indicator color, both reacted andunreacted dried samples were immersed in water for 1 day (wet) and for 1week (wet) and observed for color change. Samples treated with(N-trimethoxysilylpropyl) polyethyleneimine andN-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride showed a stablecolor using the BCIP/NBT/TRIS solution before and after reaction withthe enzyme. Results are shown in Table 21.

TABLE 20 Enzyme- Time to alkaline develop Run phosphatase color NumberTreated with Aminosilanes and GPS (units/mL) (seconds) 1 None(Untreated) 0 (Water No only) Change¹ 2 None (Untreated) 0.01 No Change¹3 None (Untreated) 0.05 No Change¹ 4 None (Untreated) 0.10 240  5 None(Untreated) 0.50 110  6 None (Untreated) 1.00 90 6(N-trimethoxysilylpropyl) 0 (Water No polyethyleneimine only) Change¹ 7(N-trimethoxysilylpropyl) 0.01 No polyethyleneimine Change¹ 8(N-trimethoxysilylpropyl) 0.05 240  polyethyleneimine 9(N-trimethoxysilylpropyl) 0.10 80 polyethyleneimine 10(N-trimethoxysilylpropyl) 0.50 25 polyethyleneimine 11(N-trimethoxysilylpropyl) 1.00 10 polyethyleneimine 123-aminopropyltriethoxysilane 0 (Water No only) Change¹ 133-aminopropyltriethoxysilane 0.01 No Change¹ 143-aminopropyltriethoxysilane 0.05 No Change¹ 153-aminopropyltriethoxysilane 0.10 90 16 3-aminopropyltriethoxysilane0.50 45 17 3-aminopropyltriethoxysilane 1.00 25 18N-trimethoxysilylpropyl-N,N,N- 0 (Water No trimethylammonium chlorideonly) Change¹ 19 N-trimethoxysilylpropyl-N,N,N- 0.01 Notrimethylammonium chloride Change¹ 20 N-trimethoxysilylpropyl-N,N,N-0.05 240  trimethylammonium chloride 21 N-trimethoxysilylpropyl-N,N,N-0.10 90 trimethylammonium chloride 22 N-trimethoxysilylpropyl-N,N,N-0.50 30 trimethylammonium chloride 23 N-trimethoxysilylpropyl-N,N,N-1.00 16 trimethylammonium chloride ¹No change within 10 minutes

TABLE 21 Color of Color of Color of Indicator Observation InitialIndicator Indicator Spot after Run Treated with Indicator Spot afterSpot after 1 week Number Aminosilanes and GPS Spot Color 1 day 1 day(wet) (wet) 24 Untreated Yellow Yellow Yellow Colorless 25 N- YellowYellow Yellow Yellow trimethoxysilylpropyl- N,N,N- trimethylammoniumchloride 26 (N- Yellow Yellow Yellow Yellow trimethoxysilylpropyl)polyethyleneimine 27 None (Untreated) Purple Purple Light Very faintPurple Purple 28 N- Purple Purple Purple Purple trimethoxysilylpropyl-N,N,N- trimethylammonium chloride 29 (N- Purple Purple Purple Purpletrimethoxysilylpropyl) polyethyleneimine

Example 30

Several 70% rayon/30% polyester nonwoven supports were treated with oneof two amine-containing silanes: (N-trimethoxysilylpropyl)polyethyleneimine and N-trimethoxysilylpropyl-N,N,N-trimethylammoniumchloride; and a wetting agent, 3-glycidoxypropyltrimethoxysilane (GPS)as described for Example 28. Additionally, one sample of the nonwovenwas left untreated. A micropipette was used to place several5-microliter drops in a spotted pattern of the 4:1:5 indicator solutionprepared as described for Example 29 onto each of the two aminosilanetreated samples and an untreated sample. These spots were allowed to airdry at room temperature for at least 30 minutes. Six (two per treatedand untreated) dried samples were used to wipe up a 10-microliter dropof alkaline phosphatase (Calbiochem) (1 unit/milliliter) on a 2.54 cm by7.62 cm (1 inch by 3 inches) glass slide. Another six (two per treatedand untreated) dried samples were dipped into ultrapure water untilthoroughly wet and used to wipe up a 20-microliter drop of alkalinephosphatase (3.5 units/milliliter) on a 2.54 cm by 7.62 cm (1 inch by 3inches) glass slide. The time needed for the first appearance of thecharacteristic purple-black color was recorded. Aminosilane-treatednonwovens exhibited a color change when used either dry or wet, and theshape of the original indicating drop was maintained. The controlsamples showed smearing of the indicator chemistry after the reactionwith the enzyme. Results are shown in Table 22.

TABLE 22 Time to develop color (seconds) Dry wipe Wet wipe with withalkaline alkaline Run Treated with phosphatase phosphatase NumberAminosilane and GPS (1 unit/mL) (3.5 units/mL) 1 None (Untreated) 540600 2 None (Untreated) 480 600 3 (N-trimethoxysilylpropyl) 35 NA¹polyethyleneimine 4 (N-trimethoxysilylpropyl) 30 50 polyethyleneimine 5N-trimethoxysilylpropyl-N,N,N- 35 60 trimethylammonium chloride 6N-trimethoxysilylpropyl-N,N,N- 25 60 trimethylammonium chloride¹Response time was not recorded.

1. A biological soil detector, comprising: A solid support membertreated with at least one amine or quaternized ammonium functionalcoating immobilizing a specific indicator; The specific indicatorimmobilized on the coating on the solid support member; wherein thespecific indicator is selected to detect a component of biological soilselected from the group consisting of protein, endotoxin, enzyme,nucleotide, and combinations of two or more of the foregoing; whereinthe specific indicator is in a predetermined or ordered pattern on thepresence of biological soil; and A retaining member holding the solidsupport thereon, the retaining member and solid support configured tofacilitate contact between the solid support a surface, wherein theretaining member is dimensioned to fit within a channel to place thesolid support member in contact with walls of the channel, and whereinthe retaining member is of sufficient length to ensure that the solidsupport member can be exposed to the walls of the channel along theentire length of the channel, the retaining member comprising a firstend and a second end and an elongate body portion extending between thefirst end and the second end; wherein the solid support member is aflexible material so that the solid support member fits within thechannel in a manner facilitating contact between the specific indicatorand the walls of the channel.
 2. The biological soil detector of claim 1wherein the solid support comprises a first material selected from thegroup consisting of at least one polymer; inorganic material; and mixedorganic and inorganic material.
 3. The biological soil detector of claim2 wherein the polymer is selected from polymers containing functionalgroups, the functional groups selected from the group consisting ofcarboxyl and salts thereof, aldehydes, sulfonic acid and salts thereof,phosphonic acid and salts thereof, alcohol, primary amine, secondaryamine, tertiary amine, amide, imide, quaternized ammonium, sulfonium,phosphonium, pyridine, cyclic amido, oxyalkylene, imidazoles andcombinations of two or more of the foregoing.
 4. The biological soildetector of claim 3 wherein the polymers containing functional groupscomprise polymers and copolymers selected from the group consisting ofcarboxyl containing polymers; polyalkoxylates; poly(meth)acrylates;polyvinyl alcohol; polyethylene-vinyl alcohol copolymer; polyurethane;polyurea; polyester; polyamide; polyimide; polyether; cellulose; rayon;polyphosphate; polypeptide; polyacrylonitrile; polyacrylamide;polycarbonate; polyethersulfone; and combinations of two or more of theforegoing.
 5. The biological soil detector of claim 2 wherein theinorganic material is selected from the group consisting of metal oxide,hydrate, metal-hydroxyl and combinations of two or more of theforegoing, and wherein the metal is Si.
 6. The biological soil detectorof claim 2 wherein the mixed organic and inorganic material is selectedfrom the group consisting of polymeric composites, ceramers, andcombinations thereof.
 7. The biological soil detector of claim 1 whereinthe solid support member comprises a first material selected from thegroup consisting of at least one polymer, inorganic material, and mixedorganic and inorganic material; and a second material attached to thefirst material.
 8. The biological soil detector of claim 7 wherein thefirst material is selected from polypropylene, polyethylene,polyvinylidene fluoride, tetrafluoroethylene hexafluoropropylenevinylidene fluoride, polyurethane, polyurea, polyester, polyvinylacetate, polyamide, polyimide, poly(meth)acrylate, polyethersulfone,glass, silica, cellulosics, rayon, polycarbonate, polyvinyl alcohol,polystyrene, and combinations of the foregoing.
 9. The biological soildetector of claim 8 wherein the second material is hydrophilic and isselected from the group consisting of monomer, polymer, reactive metalalkoxide and combinations of two or more of the foregoing.
 10. Thebiological soil detector of claim 9 wherein the second materialcomprises one or more functional groups selected from carboxyl and saltsthereof, aldehyde, sulfonic acid and salts thereof, phosphonic acid andsalts thereof, alcohol, primary amine, secondary amine, tertiary amine,amide, imide, quaternized ammonium, sulfonium, phosphonium, pyridine,cyclic amido, oxyalkylene, co-saccharinamidoundecylsiloxane, glycidyl,succinimido, imidazoles and combinations of two or more of theforegoing.
 11. The biological soil detector of claim 2 wherein the solidsupport member comprises a conformable, flexible, high integritymaterial that conforms to and fits within the inner channels of amedical device while maintaining contact with the inner surfaces of thechannel without structural failure when pushed or pulled through thelength of the channel.
 12. The biological soil detector of claim 11wherein the solied support is apolymer membrane comprises a thermallyinduced phase separation membrane comprising a material selected fromthe group consisting of high density polyethylene, polypropylene,polyvinylidenefluoride, polyethylene-vinyl copolymer and combinations oftwo or more of the foregoing.
 13. The biological soil detector of claim2 wherein the solid support member further comprises a hydrophiliccoating.
 14. The biological soil detector of claim 1 wherein thespecific indicator is selected from the group consisting of5-bromo-4-chloro-3-indolyl β-D- glucopyranoside;5-bromo-4-chloro-3-indolyl β-D-galactopyranoside;5-bromo-4-chloro-3-indolyl phosphate;5-bromo-6-chloro-3-indolyl-β-D-glucopyranoside;5-bromo-6-chloro-3-indolyl-β-D-galactopyranoside;5-bromo-6-chloro-3-indolyl phosphate;4-methylumbelliferyl-β-D-glucopyranoside;4-methylumbelliferyl-β-D-galactopyranoside;4-methylumbelliferyl-phosphate, esculin, orthophthaldialdehyde (OPA),polydiacetylenes; Bradford assay combination of compounds; Lowry assaycombination of compounds, Biuret assay combination of compounds;bicinchoninic acid (BCA); and combinations of two or more of theforegoing
 15. The biological soil detector of claim 1 wherein thespecific indicator comprises nitro blue tetrazolium chloride incombination with on or more indolyl functional indicators selected fromthe group consisting of 5-bromo-4-chloro-3-indolyl β-D -glucopyranoside;5-bromo-4-chloro-3-indolyl β-D-galactopyranoside;5-bromo-4-chloro-3-indolyl phosphate;5-bromo-6-chloro-3-indolyl-β-D-glucopyranoside;5-bromo-6-chloro-3-indolyl-β-D-galactopyranoside;5-bromo-6-chloro-3-indolyl phosphate.
 16. The biological soil indicatorof claim 15, wherein the specific indicator further comprises monovalentor divalent metal ions selected from the group consisting of sodium,potassium, zinc, manganese, magnesium, calcium and combinations of oneor more of the foregoing.
 17. The biological soil indicator of claim 1wherein the nucleotide is adenosine triphosphate.
 18. The biologicalsoil indicator of claim 1 wherein the enzyme is selected from the groupconsisting of galactosidases, phosphatases, glucosidases, andlactosidases and combinations thereof.
 19. The biological soil indicatorof claim 1 wherein specific indicator is selected to detect sulfatasesor fatty acid esterases.
 20. The biological soil detector of claim 1wherein the solid support member is releasably affixed to the retainingmember.
 21. The biological soil detector of claim 1, wherein a color ofthe specific indicator is a stable color.
 22. The biolobical soildetector of claim 1, wherein the channel is an inner channel of amedical device.
 23. The biological soil detector of claim 1, wherein themedical device is an endoscope.